Integrated monolithic microfabricated electrospray and liquid chromatography system and method

ABSTRACT

An electrospray device, a liquid chromatography device and an electrospray-liquid chromatography system are disclosed. The electrospray device comprises a substrate defining a channel between an entrance orifice on an injection surface and an exit orifice on an ejection surface, a nozzle defined by a portion recessed from the ejection surface surrounding the exit orifice, and an electrode for application of an electric potential to the substrate to optimize and generate an electrospray; and, optionally, additional electrode(s) to further modify the electrospray. The liquid chromatography device comprises a separation substrate defining an introduction channel between an entrance orifice and a reservoir and a separation channel between the reservoir and an exit orifice, the separation channel being populated with separation posts perpendicular to the fluid flow; a cover substrate bonded to the separation substrate to enclose the reservoir and the separation channel adjacent the cover substrate; and, optionally, electrode(s) for application of a electric potential to the fluid. The exit orifice of the liquid chromatography device may be homogeneously interfaced with the entrance orifice of the electrospray device to form an integrated single system. An array of multiple systems may be fabricated in a single monolithic chip for rapid sequential fluid processing and generation of electrospray for subsequent analysis, such as by positioning the exit orifices of the electrospray devices near the sampling orifice of a mass spectrometer.

FIELD OF THE INVENTION

[0001] The present invention relates generally to an integratedminiaturized chemical analysis system fabricated usingmicroelectromechanical systems (MEMS) technology. In particular, thepresent invention relates to an integrated monolithic microfabricatedelectrospray and liquid chromatography device. This achieves asignificant advantage in terms of high-throughput analysis by massspectrometry, as used, for example, in drug discovery, in comparison toa conventional system. cl BACKGROUND OF THE INVENTION

[0002] New developments in drug discovery and development are creatingnew demands on analytical techniques. For example, combinatorialchemistry is often employed to discover new lead compounds. or to createvariations of a lead compound. Combinatorial chemistry techniques cangenerate thousands or millions of compounds (combinatorial libraries) ina relatively short time (on the order of days to weeks). Testing such alarge number of compounds for biological activity in a timely andefficient manner requires high-throughput screening methods which allowrapid evaluation of the characteristics of each candidate compound.

[0003] The compounds in combinatorial libraries are often testedsimultaneously against a molecular target For example, an enzyme assayemploying a calorimetric measurement may be run in a 96-well plate. Analiquot of enzyme in each well is combined with tens or hundreds ofcompounds. An effective enzyme inhibitor will prevent development ofcolor due to the normal enzyme reaction, allowing for rapidspectroscopic (or visual) evaluation of assay results. If ten compoundsare present in each well, 960 compounds can be screened in the entireplate, and one hundred thousand compounds can be screened in 105 plates,allowing for rapid and automated testing of the compounds.

[0004] Often, however, determination of which compounds are present incertain portions of a combinatorial library is difficult, due to themanner of synthesis of the library. For example, the “split-and-pool”method of random peptide synthesis in U.S. Pat. No. 5,182,366, describesa way of creating a peptide library where each resin bead carries aunique peptide sequence. Placing ten beads in each well of a 96wellplate, followed by cleavage of the peptides from the beads and removalof the cleavage solution, would result in ten (or fewer) peptides ineach well of the plate. Enzyme assays could then be carried out in theplate wells, allowing 100,000 peptides to be screened in 105 plates.However, the identity of the peptides would not be known, requiringanalysis of the contents of each well.

[0005] The peptides could be analyzed by removing a portion of solutionfrom each well and injecting the contents into a separation device suchas liquid chromatography or capillary electrophoresis instrument coupledto a mass spectrometer. Assuming that such a method would takeapproximately 5 minutes per analysis, it would require over a month toanalyze the contents of 105 96-well plates, assuming the method wasfully automated and operating 24 hours a day.

[0006] This example illustrates the critical need for a method for rapidanalysis of large numbers of compounds or complex mixtures of compounds,particularly in the context of high-throughput screening. Techniques forgenerating large numbers of compounds, for example through combinatorialchemistry, have been established. High-throughput screening methods areunder development for a wide variety of targets, and some types ofscreens, such as the colorimetric enzyme assay described above and ELISA(enzyme linked immunosorbent assay) technology, are well-established. Asindicated in the example above, a bottleneck often occurs at the stagewhere multiple mixtures of compounds, or even multiple individualcompounds, must be characterized.

[0007] This need is further underscored when current developments inmolecular biotechnology are considered. Enormous amounts of geneticsequence data are being generated through new DNA sequencing methods.This wealth of new information is generating new insights into themechanism of disease processes. In particular, the burgeoning field ofgenomics has allowed rapid identification of new targets for drugdevelopment efforts. Determination of genetic variations betweenindividuals has opened up the possibility of targeting drugs toindividuals based on the individual's particular genetic profile.Testing for cytotoxicity, specificity, and other pharmaceuticalcharacteristics could be carried out in high-throughput assays insteadof expensive animal testing and clinical trials. Detailedcharacterization of a potential drug or lead compound early in the drugdevelopment process thus has the potential for significant savings bothin time and expense.

[0008] Development of viable screening methods for these new targetswill often depend on the availability of rapid separation and analysistechniques for analyzing the results of assays. For example, an assayfor potential toxic metabolites of a candidate drug would need toidentify both the candidate drug and the metabolites of that candidate.An assay for specificity would need to identify compounds which binddifferentially to two molecular targets such as a v iral protease and amammalian protease.

[0009] It would therefore be advantageous to provide a method forefficient proteomic screening in order to obtain the pharmacokineticprofile of a drug early in the evaluation process. An understanding ofhow a new compound is absorbed in the body and how it is metabolized canenable prediction of the likelihood for an increased therapeutic effector lack thereof.

[0010] Given the enormous number of new compounds that are beinggenerated daily, an improved system for identifying molecules ofpotential therapeutic value for drug discovery is also criticallyneeded.

[0011] It also would be desirable to provide rapid sequential analysisand identification of compounds which interact with a gene or geneproduct that plays a role in a disease of interest. Rapid sequentialanalysis can overcome the bottleneck of inefficient and time-consumingserial (one-by-one) analysis of compounds.

[0012] Accordingly, there is a critical need for high-throughputscreening and identification of compound-target reactions in order toidentify potential drug candidates.

[0013] Microchip-based separation devices have been developed for rapidanalysis of large numbers of samples. Compared to other conventionalseparation devices, these microchip-based separation devices have highersample throughput, reduced sample and reagent consumption and reducedchemical waste. The liquid flow rates for microchip-based separationdevices range from approximately 1-300 nanoliters (nL) per minute formost applications.

[0014] Examples of microchip-based separation devices include those forcapillary electrophoresis (CE), capillary electrochromatography (CEC)and high-performance liquid chromatography (HPLC). See Harrison et al,Science 1993, 261, 859-897; Jacobson et al. Anal. Chem. 1994, 66,1114-1118; and Jacobson et al. Anal. Chem. 1994, 66, 2369-2373. Suchseparation devices are capable of fast analyses and provide improvedprecision and reliability compared to other conventional analyticalinstruments.

[0015] Liquid chromatography (LC) is a well-established analyticalmethod for separating components of a fluid for subsequent analysisand/or identification. Traditionally, liquid chromatography utilizes aseparation column, such as a cylindrical tube, filled with tightlypacked beads, gel or other appropriate particulate material to provide alarge surface area. The large surface area facilitates fluidinteractions with the particulate material, and the tightly packed,random spacing of the particulate material forces the liquid to travelover a much longer effective path than the length of the column. Inparticular, the components of the fluid interact with the stationaryphase (the particles in the liquid chromatography column) as well as themobile phase (the liquid eluent flowing through the liquidchromatography column) based on the partition coefficients for each ofthe components. The partition coefficient is a defined as the ratio ofthe time an analyte spends interacting with the stationary phase to thetime spent interacting with the mobile phase. The longer an analyteinteracts with the stationary phase, the higher the partitioncoefficient and the longer the analyte is retained on the liquidchromatography column. The components may be detected spectroscopicallyafter elution from the liquid chromatography column by coupling the exitof the column to a post-column detector.

[0016] Spectroscopic detectors rely on a change in refractive index,ultraviolet and/or visible light absorption, or fluorescence afterexcitation with a suitable wavelength to detect the separatedcomponents. Alternatively, the separated components may be passed fromthe liquid chromatography column into other types of analyticalinstruments for analysis. The analysis outcome depends upon thesequenced arrival of the components separated by the liquidchromatography column and is therefore time-dependent.

[0017] The length of liquid transport from the liquid chromatographycolumn to the analysis instrument such as the detector is preferablyminimized in order to minimize diffusion and thereby maximize theseparation efficiency and analysis sensitivity. The transport length isreferred to as the dead volume or extra-column volume.

[0018] Capillary electrophoresis is a technique that utilizes theelectrophoretic nature of molecules and/or the electroosmotic flow offluids in small capillary tubes to separate components of a fluid.Typically a fused silica capillary of 100 μm inner diameter or less isfilled with a buffer solution containing an electrolyte. Each end of thecapillary is placed in a separate fluidic reservoir containing a bufferelectrolyte. A potential voltage is placed in one of the bufferreservoirs and a second potential voltage is placed in the other bufferreservoir. Positively and negatively charged species will migrate inopposite directions through the capillary under the influence of theelectric field established by the two potential voltages applied to thebuffer reservoirs. Electroosmotic flow is defined as the fluid flowalong the walls of a capillary due to the migration of charged speciesfrom the buffer solution. Some molecules exist as charged species whenin solution and will migrate through the capillary based on thecharge-to-mass ratio of the molecular species. This migration is definedas electrophoretic mobility. The electroosmotic flow and theelectrophoretic mobility of each component of a fluid determine theoverall migration for each fluidic component. The fluid flow profileresulting from electroosmotic flow is flat due to the reduction infrictional drag along the walls of the separation channel. This resultsin improved separation efficiency over liquid chromatography where theflow profile is parabolic resulting from pressure driven flow.

[0019] Capillary electrochromatography is a hybrid technique whichutilizes the electrically driven flow characteristics of electrophoreticseparation methods within capillary columns packed with a solidstationary phase typical of liquid chromatography. It couples theseparation power of reversed-phase liquid chromatography with the highefficiencies of capillary electrophoresis. Higher efficiencies areobtainable for capillary electrochromatography separations over liquidchromatography because the flow profile resulting from electroosmoticflow is flat due to the reduction in frictional drag along the walls ofthe separation channel when compared to the parabolic flow profileresulting from pressure driven flows. Furthermore, smaller particlesizes can be used in capillary electrochromatography than in liquidchromatography because no back pressure is generated by electroosmoticflow. In contrast to electrophoresis, capillary electrochromatography iscapable of separating neutral molecules due to analyte partitioningbetween the stationary and mobile phases of the column particles using aliquid chromatography separation mechanism.

[0020] The separated product of such separation devices may beintroduced as the liquid sample to a device that is used to produceelectrospray ionization. The electrospray device may be interfaced to anatmospheric pressure ionization mass spectrometer (API-MS) for analysisof the electrosprayed fluid.

[0021] A schematic of an electrospray system 50 is shown in FIG. 1. Anelectrospray is produced when a sufficient electrical potentialdifference V_(spray) is applied between a conductive or partlyconductive fluid exiting a capillary orifice and an electrode so as togenerate a concentration of electric field lines emanating from the tipor end of a capillary 52 of an electrospray device. When a positivevoltage V_(spray) is applied to the tip of the capillary relative to anextracting electrode 54, such as one provided at the ion-samplingorifice to the mass spectrometer, the electric field causespositively-charged ions in the fluid to migrate to the surface of thefluid at the tip of the capillary. When a negative voltage V_(spray) isapplied to the tip of the capillary relative to an extracting electrode54, such as one provided at the ion-sampling orifice to the massspectrometer, the electric field causes negatively-charged ions in thefluid to migrate to the surface of the fluid at the tip of thecapillary.

[0022] When the repulsion force of the solvated ions exceeds the surfacetension of the fluid sample being electrosprayed, a volume of the fluidsample is pulled into the shape of a cone, known as a Taylor cone 56which extends from the tip of the capillary. Small charged droplets 58are formed from the tip of the Taylor cone 56 and are drawn toward theextracting electrode 54. This phenomenon has been described, forexample, by Dole et al., Chem. Phys. 1968, 49. 2240 and Yamashita andFenn, J. Phys. Chem. 1984. 88, 4451. The potential voltage required toinitiate an electrospray is dependent on the surface tension of thesolution as described by, for example, Smith, IEEE Trains. Ind. Appl.1986, IA-22, 527-535. Typically, the electric field is on the order ofapproximately 10⁶ V/m. The physical size of the capillary determines thedensity of electric field lines necessary to induce electrospray.

[0023] One advantage of electrospray ionization is that the response foran analyte measured by the mass spectrometer detector is dependent onthe concentration of the analyte in the fluid and independent of thefluid flow rate. The response of an analyte in solution at a givenconcentration would be comparable using electrospray ionization combinedwith mass spectrometry at a flow rate of 100 μL/min compared to a flowrate of 100 nL/min.

[0024] The process of electrospray ionization at flow rates on the orderof nanoliters per minute has been referred to as “nanoelectrospray”.Electrospray into the ion-sampling orifice of an API mass spectrometerproduces a quantitative response from the mass spectrometer detector dueto the analyte molecules present in the liquid flowing from thecapillary.

[0025] Thus, it is desirable to provide an electrospray ionizationdevice for integration upstream with microchip-based separation devicesand for integration downstream with API-MS instruments.

[0026] Attempts have been made to manufacture an electrospray devicewhich produces nanoelectrospray. For example, Wilm and Mann, Anal. Chem.1996, 68, 1-8 describes the process of electrospray from fused silicacapillaries drawn to an inner diameter of 2-4 μm at flow rates of 20nL/min. Specifically, a nanoelectrospray at 20 nL/min was achieved froma 2 μm inner diameter and 5 μm outer diameter pulled fused-silicacapillary with 600-700 V at a distance of 1-2 mm from the ion-samplingorifice of an API mass spectrometer.

[0027] Ramsey et al., Anal. Chem. 1997, 69, 1174-1178 describesnanoelectrospray at 90 nL/min from the edge of a planar glass microchipwith a closed separation channel 10 μm deep, 60 μm wide and 33 mm inlength using electroosmotic flow and applying 4.8 kV to the fluidexiting the closed separation channel on the edge of the microchip forelectrospray formation, with the edge of the chip at a distance of 3-5mm from the ion-sampling orifice of an API mass spectrometer.Approximately 12 nL of the sample fluid collects at the edge of the chipbefore the formation of a Taylor cone and stable nanoelectrospray fromthe edge of the microchip. However, collection of approximately 12 nL ofthe sample fluid will result in remixing of the fluid, thereby undoingthe separation done in the separation channel. Remixing causes bandbroadening at the edge of the microchip, fundamentally limiting itsapplicability for nanoelectrospray-mass spectrometry for analytedetection. Thus, nanoelectrospray from the edge of this microchip deviceafter capillary electrophoresis or capillary electrochromatographyseparation is rendered impractical. Furthermore, because this deviceprovides a flat surface, and thus a relatively small amount of physicalasperity, for the formation of the electrospray, the device requires animpractically high voltage to initiate electrospray, due to poor fieldline concentration.

[0028] Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer,N. E.; Karger, B. L. Anal. Chem. 1997, 69,426-430 describes a stablenanoelectrospray from the edge of a planar glass microchip with a closedchannel 25 μm deep, 60 μm wide and 35-50 mm in length and applying 4.2kV to the fluid exiting the closed separation channel on the edge of themicrochip for electrospray formation, with the edge of the chip at adistance of 3-8 mm from the ion-sampling orifice of an API massspectrometer. A syringe pump is utilized to deliver the sample fluid tothe glass microchip electrosprayer at a flow rate between 100-200nL/min. The edge of the glass microchip is treated with a hydrophobiccoating to alleviate some of the difficulties associated withnanoelectrospray from a flat surface and which slightly improves thestability of the nanoelectrospray. Electrospraying in this manner from aflat surface again results in poor field line concentration and yieldsan inefficient electrospray.

[0029] Desai et al. 1997 International Conference on Solid-State Sensorsand Actuators, Chicago, Jun. 16-19, 1997, 927-930 describes a multi-stepprocess to generate a nozzle on the edge of a silicon microchip 1-3 μmin diameter or width and 40 μm in length and applying 4 kV to the entiremicrochip at a distance of 0.25-0.4 mm from the ion-sampling orifice ofan API mass spectrometer. This nanoelectrospray nozzle reduces the deadvolume of the sample fluid. However, the extension of the nozzle fromthe edge of the microchip exposes the nozzle to accidental breakage.Because a relatively high spray voltage was utilized and the nozzle waspositioned in very close proximity to the mass spectrometer samplingorifice, a poor field line concentration and a low efficientelectrospray were achieved.

[0030] In all of the above-described devices, edge-spraying from amonolithic chip is a poorly controlled process due to the inability torigorously and repeatably determine the physical form of the chip'sedge. In another embodiment of edge-spraying, ejection nozzles, such assmall segments of drawn capillaries, are separately and individuallyattached to the chip's edge. This process is inherently cost-inefficientand unreliable, imposes space constraints in chip design, and istherefore unsuitable for manufacturing.

[0031] Thus, it is also desirable to provide an electrospray ionizationdevice with controllable spraying and a method for producing such adevice which is easily reproducible and manufacturable in high volumes.

SUMMARY OF THE INVENTION

[0032] The present invention provides a silicon microchip-basedelectrospray device for producing reproducible, controllable and robustnanoelectrospray ionization of a liquid sample. The electrospray devicemay be interfaced downstream to an atmospheric pressure ionization massspectrometer (API-MS) for analysis of the electrosprayed fluid and/orinterfaced upstream to a miniaturized liquid phase separation device,which may have, for example, glass, plastic or silicon substrates orwafers.

[0033] The electrospray device of the present invention generallycomprises a silicon substrate or microchip defining a channel between anentrance orifice on an injection surface and a nozzle on an ejectionsurface (the major surface) such that the electrospray generated by theelectrospray device is generally approximately perpendicular to theejection surface. The nozzle has an inner and an outer diameter and isdefined by an annular portion recessed from the ejection surface. Theannular recess extends radially from the outer diameter. The tip of thenozzle is co-planar or level with and does not extend beyond theejection surface and thus the nozzle is protected against accidentalbreakage. The nozzle, channel and recessed portion are etched from thesilicon substrate by reactive-ion etching and other standardsemiconductor processing techniques.

[0034] All surfaces of the silicon substrate preferably have a layer ofsilicon dioxide thereon created by oxidization to electrically isolatethe liquid sample from the substrate and the ejection and injectionsurfaces from each other such that different potential voltages may beindividually applied to each surface and the liquid sample. The silicondioxide layer also provides for biocompatibility. The electrosprayapparatus further comprises at least one controlling electrodeelectrically contacting the substrate through the oxide layer for theapplication of an electric potential to the substrate.

[0035] Preferably, the nozzle, channel and recess are etched from thesilicon substrate by reactive-ion etching and other standardsemiconductor processing techniques. The injection-side feature(s),through-substrate fluid channel, ejection-side features, and controllingelectrodes—are formed monolithically from a monocrystalline siliconsubstrate. That is, they are formed during the course of and as a resultof a fabrication sequence that requires no manipulation or assembly ofseparate components.

[0036] Because the electrospray device is manufactured usingreactive-ion etching and other standard semiconductor processingtechniques, the dimensions of such a device can be very small, forexample, as small as 2 μm inner diameter and 5 μm outer diameter. Thus,a nozzle having, for example, 5 μm inner diameter and 250 μm in heightonly has a volume of 4.9 pL (picoliter). In contrast, an electrospraydevice from the flat edge of a glass microchip would introduceadditional dead volume of 12 nL compared to the volume of a separationchannel of 19.8 nL thereby allowing remixing of the fluid components andundoing the separation done by the separation channel. Themicrometer-scale dimensions of the electrospray device minimizes thedead volume and thereby increases efficiency and analysis sensitivity.

[0037] The electrospray device of the present invention provides for theefficient and effective formation of an electrospray. By providing anelectrospray surface from which the fluid is ejected with dimensions onthe order of micrometers, the electrospray device limits the voltagerequired to generate a Taylor cone as the voltage is dependent upon thenozzle diameter, surface tension of the fluid and the distance of thenozzle from the extracting electrode. The nozzle of the electrospraydevice provides the physical asperity on the order of micrometers onwhich a large electric field is concentrated. Further, the electrospraydevice may provide additional electrode(s) on the ejecting surface towhich electric potential(s) may be applied and controlled independent ofthe electric potentials of the fluid and the extracting electrode inorder to advantageously modify and optimize the electric field. Thecombination of the nozzle and the additional electrode(s) thus enhancethe electric field between the nozzle and the extracting electrode. Thelarge electric field, on the order of 10⁶ V/m or greater and generatedby the potential difference between the fluid and extracting electrode,is thus applied directly to the fluidic cone rather than uniformlydistributed in space.

[0038] The microchip-based electrospray ionization device of the presentinvention provides minimal extra-column dispersion as a result of areduction in the extra-column volume and provides efficient,reproducible, reliable and rugged formation of an electrospray. Thedesign of the ionization device is also robust such that theelectrospray device can be readily mass-produced in a cost-effective,high-yielding process.

[0039] In operation, a conductive or partly conductive liquid sample isintroduced into the channel through the entrance orifice on theinjection surface. The liquid sample and nozzle are held at thepotential voltage applied to the fluid, either by means of a wire withinthe fluid delivery channel to the electrospray device or by means of anelectrode formed on the injection surface isolated from the surroundingsurface region and from the substrate. The electric field strength atthe tip of the nozzle is enhanced by the application of a voltage to thesubstrate and/or the ejection surface, preferably approximately lessthan one-half of the voltage applied to the fluid. Thus, by theindependent control of the fluid/nozzle and substrate/ejection surfacevoltages, the electrospray device of the present invention allows theoptimization of the electric field lines emanating from the nozzle.Further, when the electrospray device is interfaced downstream with amass spectrometry device, the independent control of the fluid/nozzleand substrate/ejection surface voltages also allows for the directionand optimization of the electrospray into an acceptance region of themass spectrometry device.

[0040] The electrospray device of the present invention may be placed1-2 mm or up to 10 mm from the orifice of an API mass spectrometer toestablish a stable nanoelectrospray at flow rates as low as 20 nL/minwith a voltage of, for example, 700 V applied to the nozzle and 0-350 Vapplied to the substrate and/or the planar ejection surface of thesilicon microchip.

[0041] An array or matrix of multiple electrospray devices of thepresent invention may be manufactured on a single microchip as siliconfabrication using standard, well-controlled thin-film processes not onlyeliminates handling of such micro components but also allows for rapidparallel processing of functionally alike elements. The nozzles may beradially positioned about a circle having a relatively small diameternear the center of the chip. Thus, the electrospray device of thepresent invention provides significant advantages of time and costefficiency, control, and reproducibility. The low cost of theseelectrospray devices allows for one-time use such thatcross-contamination from different liquid samples may be eliminated.

[0042] The electrospray device of the present invention can beintegrated upstream with miniaturized liquid sample handling devices andintegrated downstream with an API mass spectrometer. The electrospraydevice may be chip-to-chip or wafer-to-wafer bonded to siliconmicrochip-based liquid separation devices capable of, for example,capillary electrophoresis, capillary electrochromatography, affinitychromatography, liquid chromatography (LC) or any other condensed-phaseseparation technique. The electrospray device may be alternativelybonded to glass- and/or polymer-based liquid separation devices with anysuitable method.

[0043] In another aspect of the invention, a microchip-based liquidchromatography device may be provided. The liquid chromatography devicegenerally comprises a separation substrate or wafer defining anintroduction channel between an entrance orifice and a reservoir and aseparation channel between the reservoir and an exit orifice. Theseparation channel is populated with separation posts extending from aside wall of the separation channel perpendicular to the fluid flowthough the separation channel. Preferably, the separation posts do notextend beyond and are preferably coplanar or level with the surface ofthe separation substrate such that they are protected against accidentalbreakage during the manufacturing process. Component separation occursin the separation channel where the separation posts perform the liquidchromatography function by providing large surface areas for theinteraction of fluid flowing through the separation channel. A coversubstrate may be bonded to the separation substrate to enclose thereservoir and the separation channel adjacent the cover substrate.

[0044] The liquid chromatography device may further comprise one or moreelectrodes for application of electric potentials to the fluid atlocations along the fluid path. The application of different electricpotentials along the fluid path may facilitate the fluid flow throughthe fluid path.

[0045] The introduction and separation channels, the entrance and exitorifices and the separation posts are preferably etched from a siliconsubstrate by reactive-ion etching and other standard semiconductorprocessing techniques. The separation posts are preferably oxidizedsilicon posts which may be chemically modified to optimize theinteraction of the components of the sample fluid with the stationaryseparation posts.

[0046] In another aspect of the invention, the liquid chromatographydevice may be integrated with the electrospray device such that the exitorifice of the liquid chromatography device forms a homogenous interfacewith the entrance orifice of the electro spray device, thereby allowingthe on-chip delivery of fluid from the liquid chromatography device tothe electrospray device to generate an electrospray. The nozzle, channeland recessed portion of the electrospray device may be etched from thecover substrate of the liquid chromatography device.

[0047] In yet another aspect of the invention, multiples of the liquidchromatography electrospray system may be formed on a single chip todeliver a multiplicity of samples to a common point for subsequentsequential analysis. The multiple nozzles of the electrospray devicesmay be radially positioned about a circle having a relatively smalldiameter near the center of the single chip.

[0048] The radially distributed array of electrospray nozzles on amulti-system chip may be interfaced with a sampling orifice of a massspectrometer by positioning the nozzles near the sample orifice. Thetight radial configuration of the electrospray nozzles allows thepositioning thereof in close proximity to the sampling orifice of a massspectrometer.

[0049] The multi-system chip thus provides a rapid sequential chemicalanalysis system fabricated using microelectromechanical systems (MEMS)technology. For example, the multi-system chip enables automated,sequential separation and injection of a multiplicity of samples,resulting in significantly greater analysis throughput and utilizationof the mass spectrometer instrument for, for example, high-throughputdetection of compounds for drug discovery.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The file of this patent contains at least one drawing executed incolor. Copies of this patent with color drawings will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

[0051]FIG. 1 shows a schematic of an electrospray system;

[0052]FIG. 2 shows a perspective view of an electrospray device of thepresent invention;

[0053]FIG. 3 shows a plan view of the electrospray device of FIG. 2;

[0054]FIG. 4 shows a cross-sectional view of the electrospray device ofFIG. 3 taken along line 4-4;

[0055]FIG. 5 shows a schematic of an electrospray system comprising anelectrospray device of the present invention;

[0056]FIG. 6 shows a plan view of an electrospray device having multipleelectrodes on the ejection surface of the device;

[0057]FIG. 7 shows a cross-sectional view of the electrospray device ofFIG. 6 taken along line 7-7;

[0058]FIG. 8 illustrates a feedback control circuit incorporating anelectrospray device of the present invention;

[0059] FIGS. 9-20G show an example of a fabrication sequence of theelectrospray device;

[0060]FIG. 21A shows a cross-sectional view of a piezoelectric pipettepositioned at a distance from and for delivery of a fluid sample to theentrance orifice of the electrospray device;

[0061]FIG. 21B shows a cross-sectional view of a capillary for deliveryof a fluid sample to and prior to attachment to the entrance orifice ofthe electrospray device;

[0062]FIG. 22 shows a schematic of a single integrated system comprisingan upstream fluid delivery device and an electrospray device having ahomogeneous interface with the fluid delivery device;

[0063]FIG. 23A shows an exploded perspective view of a chip-basedcombinatorial chemistry system comprising a reaction well block and adaughter plate;

[0064]FIG. 23B shows a cross-sectional view of the chip-basedcombinatorial chemistry system of FIG. 23A taken along line 23B-23B;

[0065]FIGS. 24A and 24B are color photographs of a real Taylor coneemanating from an integrated silicon chip-based nozzle;

[0066]FIGS. 24C and 24D are perspective and side cross-sectional views,respectively, of the electrospray device and mass spectrometry system ofFIGS. 24A and 24B;

[0067]FIG. 24E shows a mass spectrum of 1 μg/mL PPG425 in 50% water, 50%methanol containing 0.1% formic acid, 0.1% acetonitrile and 2 mMammonium acetate, collected at a flow rate of 333 nL/min;

[0068]FIG. 25A shows an exploded perspective view of a liquidchromatography device for homogeneous integration with the electrospraydevice of the present invention;

[0069]FIG. 25B shows a cross-sectional view of the liquid chromatographydevice of FIG. 25A taken along line 25B-25B;

[0070]FIG. 26 shows a plan view of a liquid chromatography device havingan exit orifice forming an off-chip interconnection with an off-chipdevice;

[0071]FIG. 27 shows a plan view of a liquid chromatography device havingan exit orifice forming an on-chip interconnection with another on-chipdevice;

[0072] FIGS. 28-29 show cross-sectional views of liquid chromatographydevices having alternative configurations;

[0073] FIGS. 30-35 show plan views of liquid chromatography deviceshaving alternative configurations;

[0074] FIGS. 36A-46C show an example of a fabrication sequence of theliquid chromatography device;

[0075]FIG. 47 shows a cross-sectional view of a system comprising aliquid chromatography device homogenously integrated with anelectrospray device;

[0076]FIG. 48 shows a plan view of the system of FIG. 47; and

[0077]FIG. 49 shows a detailed view of the nozzles of the system of FIG.47.

DETAILED DESCRIPTION OF THE INVENTION

[0078] An aspect of the present invention provides a siliconmicrochip-based electrospray device for producing electrosprayionization of a liquid sample. The electrospray device may be interfaceddownstream to an atmospheric pressure ionization mass spectrometer(API-MS) for analysis of the electrosprayed fluid. Another aspect of theinvention is an integrated miniaturized liquid phase separation device,which may have, for example, glass, plastic or silicon substratesintegral with the electrospray device. The descriptions that followpresent the invention in the context of a liquid chromatographseparation device. However. it will be readily recognized thatequivalent devices can be made that utilize other microchip-basedseparation devices. The following description is presented to enable anyperson skilled in the art to make and use the invention. Descriptions ofspecific applications are provided only as examples. Variousmodifications to the preferred embodiment will be readily apparent tothose skilled in the art, and the general principles defined herein maybe applied to other embodiments and applications without departing fromthe spirit and scope of the invention. Thus, the present invention isnot intended to be limited to the embodiments shown, but is to beaccorded the widest scope consistent with the principles and featuresdisclosed herein.

[0079] Electrospray Device

[0080] FIGS. 2-4 show, respectively, a perspective view, a plan view anda cross-sectional view of an electrospray device 100 of the presentinvention. The electrospray apparatus of the present invention generallycomprises a silicon substrate or microchip or wafer 102 defining achannel 104 through substrate 102 between an entrance orifice 106 on aninjection surface 108 and a nozzle 110 on an ejection surface 112. Thechannel may have any suitable cross-sectional shape such as circular orrectangular. The nozzle 110 has an inner and an outer diameter and isdefined by a recessed region 114. The region 114 is recessed from theejection surface 112, extends outwardly from the nozzle 110 and may beannular. The tip of the nozzle 110 does not extend beyond and ispreferably coplanar or level with the ejection surface 112 to therebyprotect the nozzle 110 from accidental breakage.

[0081] Preferably, the injection surface 108 is opposite the ejectionsurface 112. However, although not shown, the injection surface may beadjacent to the ejection surface such that the channel extending betweenthe entrance orifice and the nozzle makes a turn within the device. Insuch a configuration, the electrospray device would comprise twosubstrates bonded together. The first substrate may define athrough-substrate channel extending between a bonding surface and theejection surface, opposite the bonding surface. The first substrate mayfurther define an open channel recessed from the bonding surfaceextending from an orifice of the through-substrate channel and theinjection surface such that the bonding surface of the second substrateencloses the open channel upon bonding of the first and secondsubstrates. Alternatively, the second substrate may define an openchannel recessed from the bonding surface such that the bonding surfaceof the first substrate encloses the open channel upon bonding of thefirst and second substrates. In yet another variation, the firstsubstrate may further define a second through-substrate channel whilethe open channel extends between the two through-substrate channels.Thus. the injection surface is the same surface as the ejection surface.

[0082] A grid-plane region 116 of the ejection surface 112 is exteriorto the nozzle 110 and to the recessed region 114 and may provide asurface on which a layer of conductive material 119, including aconductive electrode 120, may be formed for the application of anelectric potential to the substrate 102 to modify the electric fieldpattern between the ejection surface 112, including the nozzle tip 110,and the extracting electrode 54. Alternatively, the conductive electrodemay be provided on the injection surface 108 (not shown).

[0083] The electrospray device 100 further comprises a layer of silicondioxide 118 over the surfaces of the substrate 102 through which theelectrode 120 is in contact with the substrate 102 either on theejection surface 112 or on the injection surface 108. The silicondioxide 118 formed on the walls of the channel 104 electrically isolatesa fluid therein from the silicon substrate 102 and thus allows for theindependent application and sustenance of different electricalpotentials to the fluid in the channel 104 and to the silicon substrate102. The ability to independently vary the fluid and substratepotentials allows the optimization of the electrospray throughmodification of the electric field line pattern, as described below.Alternatively, the substrate 102 can be controlled to the sameelectrical potential as the fluid when appropriate for a givenapplication.

[0084] As shown in FIG. 5, to generate an electrospray, fluid may bedelivered to the entrance orifice 106 of the electrospray device 100 by,for example, a capillary 52 or micropipette. The fluid is subjected to apotential voltage V_(fluid) via a wire (not shown) positioned in thecapillary 52 or in the channel 104 or via an electrode (not shown)provided on the injection surface 108 and isolated from the surroundingsurface region and the substrate 102. A potential voltage V_(substrate)may also be applied to the electrode 120 on the grid-plane 116, themagnitude of which is preferably adjustable for optimization of theelectrospray characteristics. The fluid flows through the channel 104and exits or is ejected from the nozzle 110 in the form of very fine,highly charged fluidic droplets 58. The electrode 54 may be held at apotential voltage V_(extract) such that the electrospray is drawn towardthe extracting electrode 54 under the influence of an electric field. Asit is the relative electric potentials which affect the electric field,the potential voltages of the fluid, the substrate and the extractingelectrode may be easily adjusted and modified to achieve the desiredelectric field. Generally, the magnitude of the electric field shouldnot exceed the dielectric breakdown strength of the surrounding medium,typically air.

[0085] In one embodiment, the nozzle 110 may be placed up to 10 mm fromthe sampling orifice of an API mass spectrometer serving as theextracting electrode 54. A potential voltage V_(fluid) ranging fromapproximately 500-1000 V, such as 700 V, is applied to the fluid. Thepotential voltage of the fluid V_(fluid) may be up to 500 V/μm ofsilicon dioxide on the surface of the substrate 102 and may depend onthe surface tension of the fluid being sprayed and the geometry of thenozzle 110. A potential voltage of the substrate V_(substrate) ofapproximately less than half of the fluid potential voltage V_(fluid),or 0-350 V, is applied to the electrode on the grid-plane 116 to enhancethe electric field strength at the tip of the nozzle 110. The extractingelectrode 54 may be held at or near ground potential V_(extract) (0 V).Thus, a nanoelectrospray of a fluid introduced to the electrospraydevice 100 at flow rates less than 1,000 nL/min is drawn toward theextracting electrode 54 under the influence of the electric field.

[0086] The nozzle 110 provides the physical asperity for concentratingthe electric field lines emanating from the nozzle 110 in order toachieve efficient electrospray. The nozzle 110 also forms a continuationof and serves as an exit orifice of the through-substrate channel 104.Furthermore, the recessed region 114 serves to physically isolate thenozzle 110 from the grid-plane region 116 of the ejection surface 112 tothereby promote the concentration of electric field lines and to provideelectrical isolation between the nozzle 110 and the grid-plane region116. The present invention allows the optimization of the electric fieldlines emanating from the nozzle 110 through independent control of thepotential voltage V_(fluid) of the fluid and nozzle 110 and thepotential voltage V_(substrate) of the electrode on the grid-plane 116of the ejection surface 112.

[0087] In addition to the electrode 120, one or more additionalconductive electrodes may be provided on the silicon dioxide layer 118on the ejection surface 112 of the substrate 102. FIGS. 6 and 7 show,respectively, a plan view and a cross-sectional view of an example of anelectrospray device 100′ wherein the conductive layer 119 defines threeadditional electrodes 122, 124, 126 on the ejection surface 112 of thesubstrate 102. Because the silicon dioxide layer 118 on the ejectionsurface 112 electrically isolates the silicon substrate 102 from theadditional electrodes 122, 124, 126 on the ejection surface 112 andbecause the additional electrodes 122, 124, 126 are physically separatedfrom each other, the electrical potential applied to each of theadditional electrodes 122, 124, 126 can be controlled independently fromeach other, from the substrate 102 and from the fluid. Thus, additionalelectrodes 122, 124, 126 may be utilized to further modify the electricfield line pattern to effect, for example, a steering and/or shaping ofthe electrospray. Although shown to be of similar sizes and shapes,electrode 120 and additional electrodes 122, 124, 126 may be of any sameor different suitable shapes and sizes.

[0088] To further control and optimize the electrospray, a feedbackcontrol circuit 130 as shown in FIG. 8 may also be provided with theelectrospray device 100. The feedback circuit 130 includes an optimalspray attribute set point 132, a comparator and voltage control 134 andone or more spray attribute sensors 136. The optimal spray attribute setpoint 132 is set by an operator or at a determined or default value. Theone or more spray attribute sensors 136 detect one or more desiredattributes of the electrospray from the electrospray device 100, such asthe electrospray ion current and/or the spatial concentration of thespray pattern. The spray attribute sensor 136 sends signals indicatingthe value of the desired attribute of the electrospray to the comparatorand voltage control 134 which compares the indicated value of thedesired attribute with the optimal spray attribute set point 132. Thecomparator and voltage control 134 then applies potential voltagesV_(fluid), V_(substrate) to the fluid and the silicon substrate 102,respectively, which may be independently varied to optimize the desiredelectrospray attribute. Although not shown, the comparator and voltagecontrol 134 may apply independently controlled additional potentialvoltages to each of one or more additional conductive electrodes.

[0089] The feedback circuit 130 may be interfaced with the electrospraydevice 100 in any suitable fashion. For example, the feedback circuit130 may be fabricated as an integrated circuit on the electrospraydevice 100, as a separate integrated circuit with electrical connectionto the electrospray device 100, or as discrete components residing on acommon substrate electrically connected to the substrate of theelectrospray device.

[0090] Dimensions of the electrospray device 100 can be determinedaccording to various factors such as the specific application, thelayout design as well as the upstream and/or downstream device to whichthe electrospray device 100 is interfaced or integrated. Further, thedimensions of the channel and nozzle may be optimized for the desiredflow rate of the fluid sample. The use of reactive-ion etchingtechniques allows for the reproducible and cost effective production ofsmall diameter nozzles, for example, a 2 μm inner diameter and 5 μmouter diameter.

[0091] In one currently preferred embodiment, the silicon substrate 102of the electrospray device 100 is approximately 250-600 μm in thicknessand the cross-sectional area of the channel 104 is less thanapproximately 50,000 μm². Where the channel 104 has a circularcross-sectional shape, the channel 104 and the nozzle 110 have an innerdiameter of up to 250 μm, more preferably up to 145 μm; the nozzle 110has an outer diameter of up to 255 μm, more preferably up to 150 μm; andnozzle 110 has a height of (and the recessed portion 114 has a depth of)up to 500 μm. The recessed portion 114 preferably extends up to 1000 μmoutwardly from the nozzle 110. The silicon dioxide layer 118 has athickness of approximately 1-4 μm, preferably 1-2 μm.

[0092] Electrospray Device Fabrication Procedure

[0093] The fabrication of the electrospray device 100 will now beexplained with reference to FIGS. 9-20B. The electrospray device 100 ispreferably fabricated as a monolithic silicon integrated circuitutilizing established, well-controlled thin-film silicon processingtechniques such as thermal oxidation, photolithography, reactive-ionetching (RIE), ion implantation, and metal deposition. Fabrication usingsuch silicon processing techniques facilitates massively parallelprocessing of similar devices, is time- and cost-efficient, allows fortighter control of critical dimensions, is easily reproducible, andresults in a wholly integral device, thereby eliminating any assemblyrequirements. Further, the fabrication sequence may be easily extendedto create physical aspects or features on the injection surface and/orejection surface of the electrospray device to facilitate interfacingand connection to a fluid delivery system or to facilitate integrationwith a fluid delivery sub-system to create a single integrated system.

[0094] Injection surface processing: entrance to through-wafer channelFIGS. 9A-11 illustrate the processing steps for the injection side ofthe substrate in fabricating the electrospray device 100 of the presentinvention. Referring to the plan and cross-sectional views.respectively, of FIGS. 9A and 9B, a double-side polished silicon wafersubstrate 200 is subjected to an elevated temperature in an oxidizingambient to grow a layer or film of silicon dioxide 202 on the injectionside 203 and a layer or film of silicon dioxide 204 on the ejection side205 of the substrate 200. Each of the resulting silicon dioxide layers202, 204 has a thickness of approximately 1-2 μm. The silicon dioxidelayers 202, 204 provide electrical isolation and also serve as masks forsubsequent selective etching of certain areas of the silicon substrate200.

[0095] A film of positive-working photoresist 206 is deposited on thesilicon dioxide layer 202 on the injection side 203 of the substrate200. An area of the photoresist 206 corresponding to the entrance to athrough-wafer channel which will be subsequently etched is selectivelyexposed through a mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

[0096] As shown in the plan and cross-sectional views, respectively, ofFIGS. 10A and 10B, after development of the photoresist 206, the exposedarea 208 of the photoresist is removed and open to the underlyingsilicon dioxide layer 202 while the unexposed areas remain protected byphotoresist 206′. The exposed area 210 of the silicon dioxide layer 202is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 206′ until thesilicon substrate 200 is reached. The remaining photoresist is removedin an oxygen plasma or in an actively oxidizing chemical bath likesulfuric acid (H₂SO₄) activated with hydrogen peroxide (H₂O₂).

[0097] As shown in the cross-sectional view of FIG. 11, an injectionside portion 212 of the through channel in the silicon substrate 200 isvertically etched by another fluorine-based etch. An advantage of thefabrication process described herein is that the dimensions of thethrough channel, such as the aspect ratio (depth to width), can bereliably and reproducibly limited and controlled. In the case where theetch aspect ratio of the processing equipment is a limiting factor, itis possible to overcome this limitation by a first etch on one side of awafer followed by a second etch on a second side of the wafer. Forexample, a current silicon etch process is generally limited to an etchaspect ratio of 30:1, such that a channel having a diameter less thanapproximately 10 μm through a substrate 200 having customary thicknessapproximately 250-600 μm would be etched from both surfaces of thesubstrate 200.

[0098] The depth of the channel portion 212 should be at or above aminimum in order to connect with another portion of the through channeletched from the ejection side 205 of the substrate 200. The desireddepth of the recessed region 114 on the ejection side 205 determinesapproximately how far the ejection side portion 220 of the channel 104is etched. The remainder of the channel 104, the injection side portion212, is etched from the injection side. The minimum depth of channelportion 212 is typically 50 μm, although the exact etch depth above theminimum etch depth does not impact the device performance or yield ofthe electrospray device.

[0099] Ejection surface processing: nozzle and surrounding surfacestructure FIGS. 12-20B illustrate the processing steps for the ejectionside 205 of the substrate 200 in fabricating the electrospray device 100of the present invention. As shown in the cross-sectional view in FIG.12, a film of positive-working photoresist 214 is deposited on thesilicon dioxide layer 204 on the ejection side 205 of the substrate 200.Patterns on the ejection side 205 are aligned to those previously formedon the injection side 203 of the substrate 200. Because silicon and itsoxide are inherently relatively transparent to light in the infraredwavelength range of the spectrum, i.e. approximately 700-1000nanometers, the extant pattern on the injection side 203 can bedistinguished with sufficient clarity by illuminating the substrate 200from the patterned injection side 203 with infrared light. Thus. themask for the ejection side 205 can be aligned within requiredtolerances.

[0100] After alignment, certain areas of the photoresist 214corresponding to the nozzle and the recessed region are selectivelyexposed through an ejection side mask by an optical lithographicexposure tool passing short-wavelength light, such as blue ornear-ultraviolet at wavelengths of 365, 405, or 436 nanometers. As shownin the plan and cross-sectional views, respectively, of FIGS. 13A and13B, the photoresist 214 is then developed to remove the exposed areasof the photoresist such that the nozzle area 216 and recessed regionarea 218 are open to the underlying silicon dioxide layer 204 while theunexposed areas remain protected by photoresist 214′. The exposed areas216, 218 of the silicon dioxide layer 204 are then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 214′ until the silicon substrate 200 isreached.

[0101] As shown in the cross-sectional view of FIG. 14, the remainingphotoresist 214′ provides additional masking during a subsequentfluorine based silicon etch to vertically etch certain patterns into theejection side 205 of the silicon substrate 200. The remainingphotoresist 214′ is then removed in an oxygen plasma or in an activelyoxidizing chemical bath like sulfuric acid (H₂SO₄) activated withhydrogen peroxide (H₂O₂).

[0102] The fluorine-based etch creates a channel 104 through the siliconsubstrate 200 by forming an ejection side portion 220 of the channel104. The fluorine based etch also creates an ejection nozzle 110, arecessed region 114 exterior to the nozzle 110 and a grid-plane region116 exterior to the nozzle 110 and to the recessed region 114. Thegrid-plane region 116 is preferably co-planar with the tip of the nozzle110 so as to physically protect the nozzle 110 from casual abrasion,stress fracture in handling and/or accidental breakage. The grid-planeregion 116 also serves as a platform on which one or more conductiveelectrodes may be provided.

[0103] The fabrication sequence confers superior mechanical stability tothe fabricated electrospray device by etching the features of theelectrospray device from a monocrystalline silicon substrate without anyneed for assembly. The fabrication sequence allows for the control ofthe nozzle height by adjusting the relative amounts of injection sideand ejection side silicon etching. Further, the lateral extent and shapeof the recessed region 114 can be controlled independently of its depth,which affects the nozzle height and which is determined by the extent ofthe etch on the ejection side of the substrate. Control of the lateralextent and shape of the recessed region 114 provides the ability tomodify and control the electric field pattern between the electrospraydevice 100 and an extracting electrode.

[0104] Oxidation for Electrical Isolation

[0105] As shown in the cross-sectional view of FIG. 15, a layer ofsilicon dioxide 221 is grown on all silicon surfaces of the substrate200 by subjecting the silicon substrate 200 to elevated temperature inan oxidizing ambient. For example, the oxidizing ambient may be anultra-pure steam produced by oxidation of hydrogen for a silicon dioxidethickness greater than approximately several hundred nanometers or pureoxygen for a silicon dioxide thickness of approximately several hundrednanometers or less. The layer of silicon dioxide 221 over all siliconsurfaces of the substrate 200 electrically isolates a fluid in thechannel from the silicon substrate 200 and permits the application andsustenance of different electrical potentials to the fluid in thechannel 104 and to the silicon substrate 200.

[0106] All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. The final thickness of the silicon dioxide can be selected toprovide the desired degree of electrical isolation in the device, wherea thicker layer of silicon dioxide provides a greater resistance toelectrical breakdown.

[0107] Metallization for Electric Field Control

[0108] FIGS. 16-20B illustrate the formation of a single conductiveelectrode electrically connected to the substrate 200 on the ejectionside 205 of the substrate 200. As shown in the cross-sectional view ofFIG. 16, a film of positive-working photoresist 222 is deposited overthe silicon dioxide layer on the ejection side 205 of the substrate 200.An area of the photoresist 222 corresponding to the electrical contactarea between the electrode and the substrate 200 is selectively exposedthrough another mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

[0109] The photoresist 222 is then developed to remove the exposed area224 of the photoresist such that the electrical contact area between theelectrode and the substrate 200 is open to the underlying silicondioxide layer 204 while the unexposed areas remain protected byphotoresist 222′. The exposed area 224 of the silicon dioxide layer 204is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 222′ until thesilicon substrate 200 is reached, as shown in the cross-sectional viewof FIG. 17.

[0110] Referring now to the cross-sectional view of FIG. 18, theremaining photoresist is then removed in an oxygen plasma or in anactively oxidizing chemical bath like sulfuric acid (H₂SO₄) activatedwith hydrogen peroxide (H₂O₂). Utilizing the patterned ejection sidesilicon dioxide layer 204 as a mask, a high-dose implantation is made toform an implanted region 225 to ensure a low-resistance electricalconnection between the electrode and the substrate 200. A conductivefilm 226 such as aluminum may be uniformly deposited on the ejectionside 205 of the substrate 200 by thermal or election-beam evaporation toform an electrode 120. The thickness of the conductive film 226 ispreferably approximately 3000 Å, although shown having a largerthickness for clarity.

[0111] The conductive film 226 may be created by any method which doesnot produce a continuous film of the conductive material on the sidewalls of the ejection nozzle 110. Such a continuous film wouldelectrically connect the fluid in the channel 104 and the substrate 200so as to prevent the independent control of their respective electricalpotentials. For example, the conductive film may be deposited by thermalor electron-beam evaporation of the conductive material, resulting inline-of-sight deposition on presented surfaces. Orienting the substrate200 such that the side walls of the ejection nozzle 110 are out of theline-of-sight of the evaporation source ensures that no conductivematerial is deposited as a continuous film on the side walls of theejection nozzle 110. Sputtering of conductive material in a plasma is anexample of a deposition technique which would result in deposition ofconductive material on all surfaces and thus is undesirable.

[0112] One or more additional conductive electrodes may be easily formedon the ejection side 205 of the substrate 200, as described above withreference to FIGS. 6 and 7. As shown in the cross-sectional view of FIG.19, a film of positive-working photoresist 228 is deposited over theconductive film 226 on the ejection side 205 of the substrate 200.Certain areas of the photoresist 228 corresponding to the physicalspaces between the electrodes are selectively exposed through anothermask by an optical lithographic exposure tool passing short-wavelengthlight, such as blue or near-ultraviolet at wavelengths of 365, 405, or436 nanometers.

[0113] Referring now to the plan and cross-sectional views of FIGS. 20Aand 20B, the photoresist 228 is developed to remove the exposed areas230 of the photoresist such that the exposed areas are open to theunderlying conductive film 226 while the unexposed areas remainprotected by photoresist 228′. The exposed areas 230 of the conductivefilm 226 are then etched using either a wet chemical etch or areactive-ion etch, as appropriate for the particular conductivematerial. The etch is either selective to the underlying silicon dioxidelayer 204 or the etch must be terminated on the basis of etch rate andtime of etch. Finally, the remaining photoresist is then removed in anoxygen plasma.

[0114] The etching of the conductive film 226 to the underlying silicondioxide layer 204 results in physically and electrically separateislands of conductive material or electrodes. As described above, theseelectrodes can be controlled independently from the silicon substrate orchannel fluid because they are electrically isolated from the substrateby the silicon dioxide and from each other by physical separation. Theycan be used to further modify the electric field line pattern andthereby effect a steering and/or shaping of the electrosprayed fluid.This step completes the processing and fabrication sequence for theelectrospray device 100.

[0115] As described above, the conductive electrode for application ofan electrical potential to the substrate of the electrospray device maybe provided on the injection surface rather than the ejection surface.The fabrication sequence is similar to that for the conductive electrodeprovided on the ejection side 205 of the substrate 200. FIGS. 20C-20Gillustrate the formation of a single conductive electrode electricallyconnected to the substrate 200 on the injection side 203 of thesubstrate 200.

[0116] As shown in the cross-sectional view of FIG. 20C, a film ofpositive-working photoresist 232 is deposited over the silicon dioxidelayer on the injection side 203 of the substrate 200. An area of thephotoresist 232 corresponding to the electrical contact area between theelectrode and the substrate 200 is selectively exposed through anothermask by an optical lithographic exposure tool passing short-wavelengthlight, such as blue or near-ultraviolet at wavelengths of 365, 405, or436 nanometers.

[0117] The photoresist 232 is then developed to remove the exposed area234 of the photoresist such that the electrical contact area between theelectrode and the substrate 200 is open to the underlying silicondioxide layer 202 while the unexposed areas remain protected byphotoresist 232′. The exposed area 234 of the silicon dioxide layer 202is then etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist 232′ until thesilicon substrate 200 is reached, as shown in the cross-sectional viewof FIG. 20D.

[0118] Referring now to the cross-sectional view of FIG. 20E, theremaining photoresist is then removed in an oxygen plasma or in anactively oxidizing chemical bath like sulfuric acid (H₂SO₄) activatedwith hydrogen peroxide (H₂O₂). Utilizing the patterned injection sidesilicon dioxide layer 202 as a mask, a high-dose implantation is made toform an implanted region 236 to ensure a low-resistance electricalconnection between the electrode and the substrate 200. A conductivefilm 238 such as aluminum may be uniformly deposited on the injectionside 203 of the substrate 200 by thermal or electron beam evaporation toform an electrode 120′.

[0119] In contrast to the formation of the conductive electrode on theejection surface of the electrospray device, sputtering, in addition tothermal or electron-beam evaporation, may be utilized to form theconductive electrode on the injection surface. Because the nozzle is onthe ejection rather than the injection side of the substrate, sputteringmay be utilized to form the electrode on the injection side as theinjection side electrode layer does not extend to the nozzle to create aphysically continuous and thus electrically conductive path with thenozzle.

[0120] With the formation of the electrode on the injection surface ofthe electrospray device, sputtering may be preferred over evaporationbecause of its greater ability to produce conformal coatings on thesidewalls of the exposed area 234 etched through the silicon dioxidelayer 202 to the substrate 200 to ensure electrical continuity andreliable electrical contact to the substrate 200.

[0121] For certain applications, it may be necessary to ensureelectrical isolation between the substrate 200 and the fluid in theelectrospray device by removing the conductive film from the region ofthe surface adjacent to the entrance orifice 106 on the injection side203. The extent of the conductive film 238 which should be removed isirrespective of etching method and may be determined by the specificmethod utilized in creating the interface between the upstream fluiddelivery system/sub-system and the injection side of the electrospraydevice. For example, a diameter of between approximately 0.2-2 mm of theconductive film 238 may be removed from the region surrounding theentrance orifice 106.

[0122] As shown in the cross-sectional view of FIG. 20F, another film ofpositive-working photoresist 240 is deposited over the conductive film238 on the injection side 203 of the substrate 200. An area of thephotoresist 240 corresponding to the region adjacent to the entranceorifice 106 on the injection side 203 is selectively exposed throughanother mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

[0123] The photoresist 240 is then developed to remove the exposed area242 of the photoresist such that the region adjacent to the entranceorifice 106 on the injection side 203 is open to the underlyingconductive film 238 while the unexposed areas remain protected byphotoresist 240′. The exposed area 242 of the conductive film 238 isthen etched by, for example, a chlorine-based plasma with a high degreeof anisotropy and selectivity to the protective photoresist 240′ untilthe silicon dioxide layer 203 is reached, as shown in thecross-sectional view of FIG. 20G.

[0124] The specific technique for etching the conductive film 238 may bedetermined by the specific conductive material deposited. For example,aluminum may be etched either in a wet chemical bath using standardaluminum etchant or in a plasma using reactive-ion etching (RIE) andchlorine-based gas chemistry. Utilization of standard wet aluminumetchant to etch an aluminum film may be preferred as such wet etchingmay facilitate the removal of any undesired conductive materialdeposited in the channel 104 via the entrance orifice 106. Further,although chlorine-based reactive-ion etching may be utilized, suchetching may lead to aluminum corrosion if removal of the photoresist isdelayed.

[0125] Forming the electrode on the injection surface for application ofan electric potential to the substrate of the electrospray device mayprovide several advantages. For example, because the ability touniformly coat photoresist on a surface is limited by nonplanar surfacetopology, coating photoresist on the much flatter injection side resultsin a more uniform and continuous photoresist film than coatingphotoresist on the ejection side. The uniformity and continuity of thephotoresist film directly and positively impact the reliability andyield, at least in part because failure of photoresist coverage wouldallow subsequent etching of silicon dioxide in undesired locationsduring the etching of exposed areas 224, 234.

[0126] Another advantage of forming the electrode on the injectionsurface is the greater flexibility and reliability in the conductivematerial deposition step because the interior surfaces of the nozzle arenot coated by the conductive material deposited onto the injectionsurface rather than onto the ejection surface of the electrospraydevice. As a result, sputtering may be utilized as a depositiontechnique to ensure conformal coating of the conductive material andelectrical continuity from the surface to the substrate contact.Further, the provision of the electrode on the injection surface doesnot preclude the deposition and patterning of additional conductiveelectrodes on the ejection side to further modify the electric fieldline pattern to effect, for example, a steering and/or shaping of theelectrospray, as such additional electrodes do not required electricalcontact to the substrate.

[0127] The ability to form the electrode on the injection surface mayalso be advantageous in certain applications where physical constraints,such as in packaging, may dictate the need for injection-side ratherthan ejection-side electrical connection.

[0128] The above described fabrication sequence for the electrospraydevice 100 can be easily adapted to and is applicable for thesimultaneous fabrication of a single monolithic system comprisingmultiple electrospray devices including multiple channels and/ormultiple ejection nozzles embodied in a single monolithic substrate.Further, the processing steps may be modified to fabricate similar ordifferent electrospray devices merely by, for example, modifying thelayout design and/or by changing the polarity of the photomask andutilizing negative-working photoresist rather than utilizingpositive-working photoresist.

[0129] Further, although the fabrication sequence is described in termsof fabricating a single electrospray device, the fabrication sequencefacilitates and allows for massively parallel processing of similardevices. The multiple electrospray devices or systems fabricated bymassively parallel processing on a single wafer may then be cut orotherwise separated into multiple devices or svstems.

[0130] Interface or Integration of the Electrospray Device

[0131] Downstream Interface or Integration of the Electrospray Device

[0132] The electrospray device 100 may be interfaced or integrateddownstream to a sampling device, depending on the particularapplication. For example, the analyte may be electrosprayed onto asurface to coat that surface or into another device for purposes ofconveyance, analysis, and/or synthesis. As described above withreference to FIG. 5, highly charged droplets are formed at atmosphericpressure by the electrospray device 100 from nanoliter-scale volumes ofan analyte. The highly charged droplets produce gas-phase ions uponsufficient evaporation of solvent molecules which may be sampled, forexample, through an orifice of an atmospheric pressure ionization massspectrometer (API-MS) for analysis of the electrosprayed fluid.

[0133] Upstream Interface or Integration of the Electrospray Device

[0134] Referring now to FIGS. 21-23, fluid may be delivered to theentrance orifice of the electrospray device in any suitable manner byupstream interface or integration with one or more fluid deliverydevices, such as piezoelectric pipettes, micropipettes, capillaries andother types of microdevices. The fluid delivery device may be a separatecomponent to form a heterogeneous interface with the entrance orifice ofthe electrospray device. Alternatively, the fluid delivery device may beintegrated with the electrospray device to form a homogeneous interfacewith the entrance orifice of the electrospray device.

[0135]FIGS. 21A and 21B illustrate examples of fluid delivery devicesforming heterogeneous interfaces with the entrance orifice of theelectrospray device. Preferably, the heterogeneous interface is anon-contacting interface where the fluid delivery device and theelectrospray device are physically separated and do not contact. Forexample, as shown in the cross-sectional view of FIG. 21A, apiezoelectric pipette 300 is positioned at a distance above theinjection surface 108 of the electrospray device 100A. The piezoelectricpipette 300 deposits a flow of microdroplets, each approximately 200 μLin volume, into the channel 104 through the entrance orifice 106A.Preferably, the electrospray device 100A provides an entrance well 302at the entrance orifice 106A for containing the sample fluid prior toentering the channel 104 particularly when it is desirable to spray avolume of fluid greater than the volume of the through-substrate channel104 and continual supply of fluid is not feasible such as when using thepiezoelectric pipette 300. The entrance well 302 preferably has a volumeof 0.1 nL to 100 nL. Furthermore, to apply an electric potential to thefluid, an entrance well electrode 304 may be provided on a surface ofthe entrance well 302 parallel to the injection surface 108.Alternatively, a wire (not shown) may be positioned in channel 104 viathe entrance orifice 106A. Preferably, some fluid is present in theentrance well 302 to ensure electrical contact between the fluid and theentrance well electrode 304.

[0136] Alternatively, the heterogeneous interface may be a contactinginterface where a fluid delivery device is attached by any suitablemethod, such as by epoxy bonding, to the electrospray device to form acontinuous sealed flow path between the upstream fluid source and thechannel of the electrospray device. For example, FIG. 21B shows across-sectional view of a capillary 306 prior to attachment to theentrance orifice 106 of the electrospray device 100B. The injectionsurface 108 of the electrospray device 100B may be adapted to facilitateattachment of the capillary 306. Such features can be easily designedinto the mask for the injection side of the substrate and can besimultaneously formed with the injection side portion of the channelduring the etching performed on the injection-side.

[0137] For example, where the inner diameter of the capillary 306 isgreater than that of the channel 104 and the entrance orifice 106, theelectrospray device 100B preferably defines a region 308 recessed fromthe injection surface 108 to form a mating collar for mating andaffixing with the capillary 306. Thus, capillary 306 may be positionedand attached in the recessed region 308 such that the exit orifice 310portion of the capillary 302 is positioned around the entrance orifice106. Further, the electrospray device 100B may optionally provide anentrance well 312 at the entrance orifice 106B for containing the samplefluid prior to entering the channel 104. Although not shown, if theouter diameter of the capillary is less than that of the channel and theentrance orifice, the capillary may be inserted into and attached to theentrance orifice of the electrospray device.

[0138] Referring now to the schematic of FIG. 22, rather than aheterogeneous interface, a single integrated system 316 is providedwherein an upstream fluid delivery device 318 forms a homogeneousinterface with the entrance orifice (not shown) of an electrospraydevice 100. The system 316 allows for the fluid exiting the upstreamfluid delivery device 318 to be delivered on-chip to the entranceorifice of the electrospray device 100 in order to generate anelectrospray.

[0139] The single integrated system 316 provides the advantage ofminimizing or eliminating extra fluid volume to reduce the risk ofundesired fluid changes, such as by reactions and/or mixing. The singleintegrated system 316 also provides the advantage of eliminating theneed for unreliable handling and attachment of components at themicroscopic level and of minimizing or eliminating fluid leakage bycontaining the fluid within one integrated system.

[0140] The upstream fluid delivery device 318 may be a monolithicintegrated circuit having an exit orifice through which a fluid samplecan pass directly or indirectly to the entrance orifice of theelectrospray device 100. The upstream fluid delivery device 318 may be asilicon microchip-based liquid separation device capable of, forexample, capillary electrophoresis, capillary electrochromatography,affinity chromatography, liquid chromatography (LC) or any othercondensed-phase separation methods. Further, the upstream fluid deliverydevice 318 may be a silicon, glass, plastic and/or polymer based devicesuch that the electrospray device 100 may be chip-to-chip orwafer-to-wafer bonded thereto by any suitable method. An example of amonolithic liquid chromatography device for utilization in, for example,the single integrated system 316, is described below.

[0141] Electrospray Device for Sample Transfer of CombinatorialChemistry Libraries Synthesized in Microdevices

[0142] The electrospray device may also serve to reproducibly distributeand deposit a sample from a mother plate to daughter plate(s) bynanoelectrospray deposition. Electrospray device(s) may be etched into amicrodevice capable of synthesizing combinatorial chemical, libraries.At the desired time, the nozzle may spray a desired amount of the samplefrom the mother plate to the daughter plate(s). Control of the nozzledimensions, applied voltages, and time of spraying may provide a preciseand reproducible method of sample deposition from an array of nozzles,such as the generation of sample plates for molecular weightdeterminations by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (MALDI-TOFMS). The capability oftransferring analytes from a mother plate to daughter plates may also beutilized to make other daughter plates for other types of assays, suchas proteomic screening.

[0143]FIGS. 23A and 23B show, respectively, an exploded perspective viewand a cross-sectional view along line 23B-23B, of a chip-basedcombinatorial chemistry system 320 comprising a reaction well block ortiter plate 322 and a receiving or daughter plate 324. The reaction wellblock 322 defines an array of reservoirs 326 for containing the reactionproducts from a combinatorially synthesized compound. The reaction wellblock 322 further defines channels 328, nozzles 330 and recessedportions 332 such that the fluid in each reservoir 326 may flow througha corresponding channel 328 and exit through a corresponding nozzle 330in the form of an electrospray. The reaction well block 322 may defineany number of reservoir(s) in any desirable configuration, eachreservoir being of a suitable dimension and shape. The volume of areservoir 326 may range from a few nanoliters up to several microlitersand more preferably ranges between approximately 200 nL to 1 μL.

[0144] The reaction well block 322 may serve as a mother plate tointerface to a microchip-based chemical synthesis apparatus such thatthe electrospray function of the reaction well block 322 may be utilizedto reproducibly distribute discreet quantities of the product solutionsto a receiving or daughter plate 324. The daughter plate 324 definesreceiving wells 334 which correspond to each of the reservoirs 326. Thedistributed product solutions in the daughter plate 324 may then beutilized to screen the combinatorial chemical library against biologicaltargets.

[0145] Illustration of an Electrospray Device Generating an ElectrospraySpray

[0146]FIGS. 24A and 24B show color images of a real Taylor coneemanating from an integrated silicon chip-based nozzle. FIGS. 24C and24D are perspective and side cross-sectional views, respectively, of theelectrospray device and mass spectrometry system shown in FIGS. 24A and24B. FIGS. 24A shows a chip-integrated electrospray device comprising anozzle and a recessed portion or annulus, and a Taylor cone, liquid jetand plume of highly-charged electrosprayed droplets of methanolcontaining 10 μg/mL polypropylene glycol 425 (PPG425) containing 0.2%formic acid. FIG. 24B shows an ion-sampling orifice of a massspectrometer in addition to the electrospray device.

[0147] The electrospray device 100 is interfaced upstream with a pipette52′. As shown in the upper right corner of each of FIGS. 24A and 24B andin FIGS. 24C and 24D, the tip of the pipette 52′ is press-sealed to theinjection side of the electrospray device 100. The electrospray device100 has a 10 μm diameter entrance orifice on the injection side, a 30 μminner diameter and a 60 μm outer diameter nozzle, a 15 μm nozzle wallthickness and a 150 μm nozzle depth. The recessed portion or the annulusextends 300 μm from the outer diameter of the nozzle. The voltageapplied to the fluid V_(fluid) introduced to the electrospray device andthus the nozzle voltage is 900 V. The voltage applied to the substrateV_(substate) and thus the electrospray device is 0 V. The voltageapplied to the mass spectrometer which also serves as an extractingelectrode V_(extract) is approximately 40 V. The liquid sample waspumped using a syringe pump at a flow of 333 nL/min through the pipettetip pressed-sealed against the injection side of the electrospraydevice. The nozzle is approximately 5 mm from the ion-sampling orifice62 of the mass spectrometer 60. The ion-sampling orifice 62 of the massspectrometer 60 generally defines the acceptance region of the massspectrometer 60. The mass spectrometer for acquiring the data was theLCT Time-Of-Flight mass spectrometer of Micromass, Inc.

[0148]FIG. 24E shows a mass spectrum of 1 μg/mL PPG425 in 50% water, 50%methanol containing 0.1% formic acid, 0.1% acetonitrile and 2 mMammonium acetate. The data were collected at a flow rate of 333 nL/min.

[0149] Liquid Chromatography Device

[0150] In another aspect of the invention shown in the explodedperspective and cross-sectional views of FIGS. 25A and 25B,respectively, a silicon-based liquid chromatography device 400 generallycomprises a silicon substrate or microchip 402 defining an introductionchannel 404 through the substrate 402 extending between an entranceorifice 406 on a first surface 408 and a fluid reservoir 410, aseparation channel 412 extending between the reservoir 410 and an exitorifice 414, a plurality of separation posts 416 along the separationchannel 412, and a cover 420 to provide an enclosure surface adjacentthe cover 420 for the reservoir 410 and the separation channel 412adjacent the cover 420.

[0151] The plurality of separation posts 416 extends from a side wall ofthe separation channel 412 in a direction perpendicular to the fluidflow though the separation channel 412. Preferably, one of the ends ofeach separation post 416 does not extend beyond and is preferablycoplanar or level with the second surface 417. The separation channel412 is functionally similar to the liquid chromatography column in thatcomponent separation occurs in the separation channel 412 where theplurality of separation posts 416 perform the liquid chromatographyfunction. Component separation occurs through the interaction of thefluid flowing through the separation channel 412 wherein the columnarseparation posts 416 provides the large surface area. The surfaces ofthe separation channel 412 and the separation posts 416 are preferablyprovided with an insulating layer to insulate the fluid in theseparation channel 412 from the substrate 402. Specifically, theseparation posts 416 are preferably oxidized silicon posts which may bechemically modified using known techniques in order to optimize theinteraction of the components of the sample fluid with the stationaryphase, the separation posts 416. In one embodiment, the separationchannel 412 extends beyond the separation posts 416 to the edge of thesubstrate 402 and terminating as the exit orifice 414.

[0152] The introduction channel, 404, the separation channel 412, thereservoir 410 and the separation posts 416 may have any suitablecross-sectional shapes such as circular and/or rectangular. Preferably,the separation posts 416 have the same cross-sectional shapes and sizesbut may nonetheless have different cross-sectional shapes and/or sizes.

[0153] The liquid chromatography device 400 further comprises a layer ofsilicon dioxide 422 over the surfaces of the substrate of the cover 420and a layer of silicon dioxide 424 over the surfaces of the substrate402. The silicon dioxide layers 422, 424 electrically isolate a fluidcontained in the reservoir 410 and the separation channel 412 from thesubstrate 402 and the substrate of the cover 420. The silicon dioxidelayers 422, 424 are also relatively inactive and thus less likely tointeract with fluids in the reservoir 410 and the separation channel 412than bare silicon.

[0154] Depending on the specific application, the substrate 402 mayprovide a surface on which one or more conductive electrodes inelectrical contact with the fluid in the device 400 may be formed. Forexample, a reservoir electrode 426 and/or an exit electrode 428 may beprovided on the second surface 417 of the substrate 402 such that acorresponding electrode would be in electrical contact with fluid in thereservoir 410 and near the exit orifice 414, respectively. A fillingelectrode 430 may also be provided on the second surface 417 of thesubstrate 402 such that it would be in electrical contact with fluid inthe unpopulated portion 432 of the separation channel 412 between thereservoir 410 and the first occurrence of separation posts 416. Theshape, size and location along the fluidic flow path of each electrodeon the substrate 402 may be determined by design considerations such asthe distance between adjacent electrodes. Further, any or all of theelectrodes may be alternatively or additionally formed on the bondingsurface 425 of the cover 420. For example, the filling electrode 430 maybe alternatively positioned such that it would be in electrical contactwith fluid in the separation channel 412 adjacent the reservoir 410.Further, additional electrodes may be provided, for example, to createan arbitrary electrical potential distribution along the fluidic flowpath.

[0155] Providing two or more of the reservoir, filling and exitelectrodes along with electrical isolation of the fluid sample in thedevice 400 from the substrate 402 and the substrate of the cover 420allows for the application and sustenance of different (or same)electric potentials at two or more different locations along the fluidicpath. The difference in electric potentials at two or more differentlocations along the fluidic path causes fluidic motion to occur betweenthe two or more locations. Thus, these electrodes may facilitate thefilling of the reservoir 410 and/or the driving of the fluid through theseparation channel 412.

[0156] Further, through appropriate layout design and fabricationprocesses, the substrate 402 and/or the cover 420 may also provideadditional functionalities such as pre-conditioning of the fluid priorto delivery into the reservoir 410, and/or conveying, analyzing, and/orotherwise treating fluidic samples exiting from the separation channel412. The cover 420 may provide such additional functionality on eitheror both surfaces and/or the bulk of the cover 420.

[0157] The cover 420 may comprise a substrate 418 comprising silicon orany other suitable material, such as glass. plastics and/or polvmers.The specific material for the cover 420 may depend upon, for example,whether direct observation of a fluoresced fluid is desired such thatglass may be more desirable and/or the consideration of the ease offabrication of the cover 420 by utilizing similar processing techniquesas for the substrate 402 such that silicon may be more desirable. Thecover 420 may be bonded or otherwise affixed to form a hermetic sealbetween the substrate 402 and the cover 420 in order to ensure theappropriate level of fluid containment and isolation. For example,several methods of bonding silicon to silicon or glass to silicon areknown in the art, including anodic bonding, sodium silicate bonding,eutectic bonding, and fusion bonding. The specific hermetic bondingmethod may depend on various factors such as the physical form of thesurfaces of the substrate 402 and the cover 420 and/or the applicationand functionality of the integrated system and/or the liquidchromatography device 400.

[0158] Dimensions of the liquid chromatography device 400 may bedetermined according to various factors such as the specificapplication, the layout design as well as the device with which it is tobe interfaced or integrated. The surface dimensions, i.e. the dimensionsin the X and Y directions, of the elements of the liquid chromatographydevice 400 may be determined by layout design and through thecorresponding photomasks used in fabrication. The depth or height, i.e.the dimension in the Z direction, of the elements of the liquidchromatography device 400 may be determined by the etch processes duringfabrication, as described below. The depth or height of the elements isindependent of the surface dimensions to a first-order approximationalthough the aspect ratio limitations of the reactive-ion etch placesconstraints on the etch depth, particularly with the small surfaceopenings in the channel 412 between the separation posts 416.

[0159] Further, the size, number, cross-sectional shape, spacing andplacement of the separation posts 416 may also be determined by layoutdesign to achieve the desired flow rate and to prevent low-resistancelines of sight within the separation channel 412 to ensure adequatefluid-surface interaction. Each separation post 416 may have the same ordifferent characteristics such as size and/or cross-sectional shape. Thecross-sectional shape of the posts may be chosen in layout design tooptimize fluid/boundary layer interactions at the post surfaces. Theseparation posts 416 may be placed in any desired pattern in theseparation channel 412, such as periodic, semiperiodic, or random. Closespacing of the separation posts 416 may be desirable for maximization ofthe surface interactions with the fluid. Similarly, minimizing thecross-sectional area of the separation posts 416 may permit placement ofgreater number in the separation channel 412. However, the reduction ofthe cross-sectional area of the separation posts 416 is limited by theresulting reduction in the mechanical stability necessary duringprocessing.

[0160] Control of the size, number, cross-sectional shape, spacing andplacement of the separation posts 416 provides advantages overtraditional liquid chromatography as the traditional separation columnpacking materials have undesired dispersion in size distribution as wellas random spacing variations.

[0161] In one currently preferred embodiment, the substrate 402 of theliquid chromatography device 400 is approximately 250-600 μm inthickness, the separation channel 412 has a depth of approximately 10μm, the rectangular reservoir 410 is approximately 1000 μm by 1000 μmresulting in a volume of approximately 10 nL. The depth of the reservoir410 and the separation channel 412 is limited by the height of theseparation posts 416 which is in turn limited by the maximum etch aspectratio. The nearest-neighbor spacing of the separation posts 416 ispreferably less than approximately 5 μm. The dimensions of the reservoir410 determine the volume of the fluid sample which can be used for theliquid chromatography separation and, as is evident, through theindependent control of surface dimensions and the depth, the reservoir410 may be designed to have any desired volume. Preferably, the diameterof the entrance orifice 406 is 100 μm or less such that the fluidsurface tension would be sufficient to maintain the fluid in thereservoir 410 to prevent leakage therefrom.

[0162] The silicon-based liquid chromatography device 400 reduces thesize of a typical liquid chromatography device by nearly two orders ofmagnitude. The dimensional scaling may provide the advantage ofsignificantly reducing the mass of the analyte and/or the volume of thefluid sample required for accurate analysis. Further, by reducing amacroscopic separation column and its packing materials to a monolithicdevice, the liquid chromatography device 400 can be a component of anon-chip integrated system.

[0163] Further, all features such as the reservoir, the separationchannel and the separation posts are recessed from the substrate 402.The portion of the substrate 402 exterior to the reservoir and theseparation channel thus serves to physically protect the separationposts from casual abrasion and stress fracture in handling andsubsequent bonding of the substrate 402 and the cover 420. Because theposts are integral with the substrate, the posts are inherently stableand thus allow for the use of a pressurized system without the risk ofdamage to the stationary phase which may otherwise result with the useof conventional packing materials in conventional high-performanceliquid chromatography systems.

[0164] An upstream fluid delivery system, such as a micropipette,piezoelectric pipette or small capillary, may be press-sealed onto theexterior surface of the liquid chromatography device 400 such that thepipette or capillary is concentric with the entrance orifice 406.Optionally, the liquid chromatography device may provide a collar (notshown) to facilitate the mating and affixing of the fluid deliver deviceto the liquid chromatography device, similar to the mating collar of theelectrospray device as discussed with reference to FIG. 21B.

[0165] To operate the liquid chromatography device 400, the fluidreservoir 410 may first be filled with a sample fluid by injecting thefluid from a fluid delivery device through the introduction channel 404via the entrance orifice 406. Any suitable fluid delivery device such asa micropipette, a piezoelectric pipette or a small capillary may beutilized. The volume of the sample fluid injected into the liquidchromatography device 400 may be up to approximately the volume of thereservoir 410 plus a relatively small volume remaining in theintroduction channel 404.

[0166] The filling of the reservoir 410 may be facilitated by applyingan appropriate potential voltage difference between the reservoirelectrode 426 and the filling electrode 430, such as approximately 1000V/cm of introduction channel 404. In particular, a volume of the fluidis first introduced into the reservoir 410 through the introductionchannel 404 via the entrance orifice 406 to coat or prime the surfacesof the reservoir 410 and the introduction channel 404 by capillaryaction to allow for electrical contact between the fluid and thereservoir and filling electrodes 426, 430. Where the filling electrode430 is positioned in a portion of the separation channel 412 unpopulatedby separation posts 416, the filling electrode 430 also facilitates thefilling of the portion of the channel 412 between the reservoir 410 andthe filling electrode 430.

[0167] After filling the reservoir 410 with an appropriate volume of thesample fluid, any suitable method may then be utilized to drive thefluid from the reservoir 410 into the separation channel 412. Forexample, the fluid may be driven from the filled reservoir 410 throughthe separation channel 412 by applying hydrostatic pressure to thereservoir 410 via the entrance orifice 406.

[0168] Alternatively or additionally, the fluid may be driven throughthe separation channel 412 by applying a suitable electrokineticpotential voltage difference between the reservoir electrode 426 and theexit electrode 428 to generate electrophoretic or electroosmotic fluidicmotion. Preferably, the electric potential difference is approximately1000 V/cm of separation channel length. Of course, any other suitablemethods of inducing fluidic motion may be utilized. Pressure-driven andvoltage-driven flow effect different separation efficiencies. Thus,depending upon the application, one or both may be utilized.

[0169] Fluid then exits from the separation channel 412 through the exitorifice 414 to, for example, a capillary 434, which has an off-chipinterconnection with the exit orifice 414, as shown in FIG. 26.Alternatively, as shown in FIG. 27, the liquid chromatography device 400may perform separation on the fluid from reservoir 410 such thatselected analytes from the separation performed by posts 416 passesthrough unpopulated channel 436 to another on-chip device 438, such asfor analysis and/or mixing, while the remainder of the fluid is directedto the waste reservoir 439. The unpopulated channel 436 may be a merecontinuation of the separation channel 412 of the liquid chromatographydevice 400 or a channel separate from the separation channel 412.

[0170] Two or more fluid samples may be driven through the liquidchromatography device 400 by successively filling the reservoir anddriving the fluid through the separation channel 412. For example, incertain applications, it may be desirable or necessary to first coat thesurfaces of the separation posts 416 with one or more reagents and thenpass an analyte sample over the conditioned separation posts 416.

[0171] Various modifications may be made to the liquid chromatographydevice describe above. For example, as shown in FIG. 28, rather thandefining the entrance orifice and the introduction channel in thesubstrate, the liquid chromatography device 400′ may provide anintroduction channel 404′ in the cover 420′ such that the entranceorifice 406′ is defined on an exterior surface of the cover 420′.Further, the cover 420′ may define an exit channel 413 between an exitorifice 414′ defined on an exterior surface of the cover 420′ and aseparation channel 412′ which terminates within the substrate 402′.

[0172] In another variation, an additional introduction channel 440 andentrance orifice 442 may be defined in the substrate 402″, as shown inFIG. 29, or in the cover (not shown). The additional introductionchannel 440 introduces fluid to the separation channel 412″ such thatthe fluid from the additional introduction channel 440 intersects thepath of fluid flow from the reservoir 410 through the unpopulatedportion 432″ of the separation channel 412″. The fluid reservoir 410 maybe utilized as a buffer for an eluent and the additional introductionchannel 440 may be utilized to introduce the fluid sample to theseparation channel 412″. Further, the additional entrance orifice 442may be utilized to introduce several fluid samples in succession intothe separation channel 412″. For example, in certain applications, itmay be necessary to first coat the surfaces of the separation posts 416with one reagent and then pass an analyte over the conditioned surfacesof the separation posts 416.

[0173] Referring now to FIGS. 30-35, although the liquid chromatographydevice has been described as comprising a single reservoir and a singleseparation channel, the monolithic liquid chromatography device may beeasilv adapted and modified to comprise multiples of the liquidchromatography device and/or multiple entrance orifices, exit orifices,reservoirs and/or separation channels. In each of the variations, any orall of the reservoir(s), separation channel(s), and separation posts mayhave different dimensions and/or shapes.

[0174] For example, multiple reservoir-separation channel combinationsmay be provided on a single chip. In particular, as shown in FIG. 30, areservoir 410A may feed into a separation channel 412A having separationposts 416A and another reservoir 410B may feed into another separationchannel 412B having separation posts 416B.

[0175] In another variation as shown in FIG. 31, a single reservoir 410Cmay feed multiple separation channels 412C, 412D. Each of separationchannels 412C, 412D may have therein separation posts 416C, 416D,respectively, which may have the same or different properties, such asnumber, size and shape. Another channel 412E may be provided as a nullchannel completely unpopulated by separation posts. The output from thenull channel 412E may be utilized as a basis of comparison to the outputfrom the separation channel(s) populated by separation posts.Alternatively, all of the channels 412C, 412D, 412E may be separationchannels having separation posts.

[0176] Referring now to FIG. 32, fluid from multiple reservoirs 410E and410F may feed into a single separation channel 412F via connectingchannels 444E, 444F, respectively. The connecting channels 444E, 444Fare preferably unpopulated by separation posts to facilitate the mixingof the fluid samples from the reservoirs 410E, 410F prior to passagethrough the separation channel 412F. The mixing of samples may beutilized to condition the primary sample of interest prior to separationor to effect a reaction between the samples prior to passage through thepopulated portion of the separation channel 412F. Alternatively, fluidsuch as a conditioning fluid from one reservoir 410E may flow throughthe separation channel 412F in order to condition the surfaces of theseparation posts 416F prior to the passage of the other sample such asan analyte sample from the other reservoir 410F. Although the separationposts 416F are shown as having different cross-sections, separationposts 416F may have the same size and cross-sectional shape.

[0177] Alternatively, in addition to having fluid from multiplereservoirs feed into a single separation channel via connectingchannels, fluid from another reservoir may be introduced to the fluidflow along the separation channel, before and/or after the fluid haspassed through the populated portion of the separation channel. Forexample, FIG. 33 shows that the fluid from multiple reservoirs 410G,410H may be fed into a single separation channel 412G via connectingchannels 444G, 444H, respectively, and fluid from another reservoir 4101may be introduced to the fluid flow along the separation channel 412Gafter the fluid has passed the separation posts 416G. FIG. 34 shows thatthe fluid from multiple reservoirs 410J, 410K may be fed into a singleseparation channel 412J via connecting channels 444J, 444K,respectively, and fluid from another reservoir 410L may be introduced tothe fluid flow along the separation channel 412J prior to the fluidpassing the separation posts 416J.

[0178] For devices having multiple reservoirs and/or multiple channels,separate electrodes may be provided for each reservoir and/or for eachchannel, for example, in the unpopulated portion of the channel upstreamfrom the separation posts and/or near the exit of the channel. Suchprovision of separate electrodes allow for the separate and independentcontrol of the fluidic flow for filling each reservoir and/or fordriving the fluid through the separation channel.

[0179] The electric control may be simplified by having one commonreservoir electrode, one common filling electrode, and/or one exitelectrode among the multiple reservoirs and/or multiple channels. Forexample, each of the multiple reservoirs may be separately filled byapplying a first voltage to the common reservoir electrode and a secondvoltage, different from the first voltage, to the filling electrodecorresponding to the reservoir to be filled while applying the firstvoltage to each of the other filling electrodes. As is evident, themultiple reservoirs may be simultaneously filled by applying a firstvoltage to the common reservoir electrode and a second, differentvoltage to each of the filling electrodes. Similarly, fluid may beseparately driven through each of the multiple channels by applying athird voltage to the common reservoir electrode while applying a fourthvoltage, different from the third voltage, to the exit electrodecorresponding to the channel through which fluid is to be driven and thethird voltage to each of the other exit electrodes.

[0180] In yet another variation shown in FIG. 35, in addition to asample reservoir 410M and separation posts 416M, a plurality of posts416L may be provided in a channel 412M upstream from the separationposts 416M for providing additional functionality such as solid-phaseextraction (SPE) for sample pretreatment. The SPE posts 416L may be thesame, similar to or different from the separation posts 416M simply byvarying the layout design. The SPE posts 416L may provide surfacefunctionality different from that of the separation posts 416M.Alternatively, rather than providing a sample reservoir, an introductionchannel (not shown) may be utilized to introduce a fluidic sampledirectly in the channel 412M by allowing direct injection of the sampletherein. Further, reservoirs 410N, 410P may be provided to containfluidic buffers necessary for sample pretreatment upstream of the posts416L. For example, an eluent reservoir may be provided for elutinganalytes and a wash reservoir may be provided for sample cleanup.

[0181] After the fluid samples pass the SPE posts 416L, waste productsfrom, for example, the solid-phase extraction process may be directedinto a waste reservoir 410Q. In particular, during the SPE process,voltage differences may be applied between or amongst reservoirs 410M,410N, 410P, and 410Q such that a portion of the fluid from reservoirs410M, 410N is directed to waste reservoir 410Q while the remainingportion of the fluid from reservoir 410M remain on the SPE posts 416L.Material may then be washed off of the SPE posts 416L by directing fluidfrom, for example, reservoir 410P through channel 412M for separation ofthe extracted material by separation posts 416M. Additional reservoirs410R, 410S downstream of the waste reservoir 410Q and upstream of theseparation posts 416M may be provided to contain gradient elution ofanalytes in one reservoir and a diluent in the other reservoir. Gradientelution facilitates chromatography by changing the mobile phasecomposition, i.e. the polarity to facilitate analyte interactions withthe stationary phase, and thus facilitate separation of the analytes. Inaddition, the diluent provides the correct polarity of the solution forthe next separation.

[0182] Liquid Chromatography Device Fabrication Procedure

[0183] The fabrication of the liquid chromatography device of thepresent invention will now be explained with reference to FIGS. 36A-46B.The liquid chromatography device is preferably fabricated as amonolithic silicon micro device utilizing established, well-controlledthin-film silicon processing techniques such as thermal oxidation,photolithography, reactive-ion etching (RIE), ion implantation, andmetal deposition. Fabrication using such silicon processing techniquesfacilitates massively parallel processing of similar devices, is time-and cost-efficient, allows for tighter control of critical dimensions,is easily reproducible, and results in a wholly integral device, therebyeliminating any assembly requirements. Manipulation of separatecomponents and/or sub-assemblies to build an liquid chromatographydevice with high reliability and yield is not desirable and may not bepossible at the micrometer dimensions required for efficient separation.

[0184] Further, the fabrication sequence may be easily extended tocreate physical aspects or features to facilitate interfacingintegration and/or connection with devices having other functionalitiesor to facilitate integration with a fluid delivery subsystem to create asingle integrated system. Consequently, the liquid chromatography devicemay be fabricated and utilized as a disposable device, therebyeliminating the need for column regeneration and eliminating the risksof sample crosscontamination.

[0185] Referring to the plan and cross-sectional views, respectively, ofFIGS. 36A and 36B, a silicon wafer separation substrate 500, double-sidepolished and approximately 250-600 μm in thickness, is subjected to anelevated temperature in an oxidizing ambient to grow a layer or film ofsilicon dioxide 502 on the reservoir side 503 and a layer or film ofsilicon dioxide 504 on the back side 505 of the separation substrate500. Each of the resulting silicon dioxide layers 502, 504 has athickness of approximately 1-2 μm. The silicon dioxide layers 502, 504provide electrical isolation and also serve as masks for subsequentselective etching of certain areas of the separation substrate 500.

[0186] A film of positive-working photoresist 506 is deposited on thesilicon dioxide layer 502 on the reservoir side 503 of the separationsubstrate 500. Certain areas of the photoresist 506 corresponding to thereservoir, separation channel and separation posts which will besubsequently etched are selectively exposed through a mask by an opticallithographic exposure tool passing short-wavelength light, such as blueor near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.

[0187] Referring to the plan and cross-sectional views, respectively, ofFIGS. 37A and 37B, after development of the photoresist 506, the exposedareas 508, 509, 510 of the photoresist corresponding to the reservoir,separation posts and channel, respectively, are removed and open to theunderlying silicon dioxide layer 502 while the unexposed areas remainprotected by photoresist 506′. The exposed areas 508, 509, 510 of thesilicon dioxide layer 502 are then etched by a fluorine-based plasmawith a high degree of anisotropy and selectivity to the protectivephotoresist 506′ until the silicon separation substrate 500 is reached.The remaining photoresist is removed in an oxygen plasma or in anactively oxidizing chemical bath like sulfuric acid (H₂SO₄) activatedwith hydrogen peroxide (H₂O₂).

[0188] As shown in the cross-sectional view of FIG. 38, the reservoir410, the separation channel 412, and the separation posts 416 in theseparation channel 412 are vertically formed in the silicon separationsubstrate 500 by another fluorine-based etch. Preferably, the reservoir410 and the separation channel 412 have the same depth controlled by theetch time at a known etch rate. The simultaneous formation of thereservoir 410 and the channel 412 ensures uniform depth such that thereare no discontinuities in the fluid-constraining surfaces to impede thefluid flow. The depth of the reservoir 410 and the channel 412 ispreferably between approximately 5-20 μm and more preferablyapproximately 10 μm. The etch can reliably and reproducibly be executedto produce an aspect ratio (etch depth to width) of up to 30:1. Althoughnot shown, any other reservoirs and/or channels, populated orunpopulated, may also be formed by this etch sequence.

[0189] A film of positive-working photoresist is then deposited over thesilicon dioxide layer 502 and the exposed separation substrate 500 onthe reservoir side 503 of the separation substrate 500. An area of thephotoresist corresponding to the introduction channel which will besubsequently etched is selectively exposed through a mask by an opticallithographic exposure tool passing short-wavelength light, such as blueor near-ultraviolet at wavelengths of 365, 405, or 436 nanometers. Afterdevelopment of the photoresist, the exposed area of the photoresistcorresponding to the introduction channel is removed and open to theunderlying separation substrate 500 while the unexposed areas remainprotected by the photoresist.

[0190] As shown in the plan and cross-sectional views of FIGS. 39A and39B, respectively, the exposed area of the separation substrate 500 isthen vertically etched by a fluorine-based plasma with a high degree ofanisotropy and selectivity to the protective photoresist until thesilicon dioxide layer 504 on back side 505 is reached. Thus, a portionof the introduction channel 404 is formed through the separationsubstrate 500. The remaining photoresist is removed in an oxygen plasmaor in an actively oxidizing chemical bath like sulfuric acid (H₂SO₄)activated with hydrogen peroxide (H₂O₂). The silicon dioxide layer 504on the back side 505 may then be removed by, for example, an unpatternedetch in a fluorine-based plasma.

[0191] Alternatively, as shown in FIGS. 40A and 40B, the introductionchannel 404 may be formed by etching from both the reservoir side 503and the back side 505 of the substrate 500. After performing a verticaletch though a portion of the substrate 500 to form a portion of theintroduction channel 404 in a manner similar to that described above, afilm of positive-working photoresist 512 is deposited on the silicondioxide layer 504 on the back side 505 of the separation substrate 500.Patterns on the back side 505 may be aligned to those previously formedon the reservoir side 503 of the separation substrate 500. Becausesilicon and its oxide are inherently relatively transparent to light inthe infrared wavelength range of the spectrum, i.e. approximately700-1000 nanometers, the extant pattern on the reservoir side 503 can bedistinguished with sufficient clarity by illuminating the separationsubstrate 500 from the patterned reservoir side 503 with infrared light.Thus, the mask for the back side 505 can be aligned within requiredtolerances. Upon alignment, an area of the photoresist 512 correspondingto the entrance orifice and the introduction channel which will besubsequently etched is selectively exposed through a mask by an opticallithographic exposure tool passing short-wavelength light, such as blueor near-ultraviolet at wavelengths of 365, 405, or 436 nanometers.

[0192] After development of the photoresist 512, the exposed area 514 ofthe photoresist corresponding to the entrance orifice is removed toexpose the underlying silicon dioxide layer 504 on the back side 505 ofthe separation substrate 500 while the unexposed areas remain protectedby the photoresist 512. The exposed area 514 of the silicon dioxidelayer 504 is then etched by a fluorine-based plasma with a high degreeof anisotropy and selectivity to the protective photoresist 512 untilthe substrate 500 is reached. The remaining photoresist providesadditional masking during a subsequent fluorine-based silicon etch tovertically etch the backside portion of the introduction channel. Thus,a through-substrate introduction channel 404 is complete. The remainingphotoresist is removed in an oxygen plasma or in an actively oxidizingchemical bath like sulfuric acid (H₂SO₄) activated with hydrogenperoxide (H₂O₂).

[0193] Preferably, the introduction channel 404 has the same diameter asthe entrance orifice. A practical limit on etch aspect ratio of 30:1constrains the diameter of the entrance orifice being etched to beapproximately 10 μm or greater for substrates of approximately 300 μmthickness. Preferably, the entrance orifice 406 and the introductionchannel 404 are approximately 100 μm in diameter due to practicalconsiderations. For example, the etch aspect ratio imposes a minimumdiameter, and the diameter is preferably sufficiently large to enableease of filling the reservoir 410 yet sufficiently small to ensure afluid surface tension to prevent the fluid from leaking out of thereservoir 410.

[0194] Alternatively, both the introduction channel and the entranceorifice may be formed by etching from the back side 505 of theseparation substrate 500. This may be preferable as it may be difficultto satisfactorily coat the separation posts 416 with photoresist.Further, this may be desirable depending on the application of thedevice, e.g. the external sample delivery system, the desired chiphandling devices, the interfacing with other devices, chip-based ornon-chip based, and/or the packaging considerations of the chip.Referring to the cross-sectional view of FIG. 41, after the reservoir,separation channel and the separation posts are etched in the separationsubstrate 500 (shown in FIG. 38), a film of positive-working photoresist516 is deposited on the silicon dioxide layer 504 on the back side 505of the separation substrate 500. Patterns on the back side 505 may bealigned to those previously formed on the reservoir side 503 of theseparation substrate 500 by illuminating the separation substrate 500from the patterned reservoir side 503 with infrared light, as describedabove. Upon alignment, an area of the photoresist 516 corresponding tothe entrance orifice which will be subsequently etched is selectivelyexposed through a mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

[0195] After development of the photoresist 516, the exposed area 518 ofthe photoresist 516 corresponding to the entrance orifice is removed toexpose the underlying silicon dioxide layer 504 on the back side 505 ofthe separation substrate 500. The exposed area 518 of the silicondioxide layer 504 is then etched by a fluorine-based plasma with a highdegree of anisotropy and selectivity to the protective photoresist 512until the silicon separation substrate 500 is reached. The remainingphotoresist is left in place to provide additional masking during thesubsequent etch through the silicon separation substrate 500.

[0196] Referring now to the cross-sectional view of FIG. 42, theintroduction channel 404 is vertically formed through the siliconseparation substrate 500 by another fluorine-based etch. Theintroduction channel 404 is completed by etching through the separationsubstrate 500 until the reservoir 410 is reached. Thus, the introductionchannel 404 extends through the separation substrate 500 between theentrance orifice 406 on the back side 505 of the separation substrate500 and the reservoir 410. The remaining photoresist is removed in anoxygen plasma or in an actively oxidizing chemical bath like sulfuricacid (H₂SO₄) activated with hydrogen peroxide (H₂O₂).

[0197] Oxidation for Surface Passivation and Fluid Isolation

[0198] As shown in the cross-sectional view of FIG. 43, a layer ofsilicon dioxide 522 is grown on all silicon surfaces of the substrate500 by subjecting the silicon substrate 500 to elevated temperature inan oxidizino ambient. For example, the oxidizing ambient may be anultra-pure steam produced by oxidation of hydrogen for a silicon dioxidethickness greater than approximately several hundred nanometers or pureoxygen for a silicon dioxide thickness of approximately several hundrednanometers or less. The layer of silicon dioxide 522 over all siliconsurfaces of the separation substrate 500 electrically isolates a fluidin the channel from the silicon substrate 500 and permits theapplication and sustenance of an electric potential difference betweenthe reservoir and the exit of the separation channel, between thereservoir and an unpopulated portion of the separation channel near thereservoir to facilitate in filling the reservoir and/or between otherpoints along the fluid flow path. Thus, the application and sustenanceof a significant voltage across the fluid sample may be achieved.Further, oxidation renders a surface inactive relative to a bare siliconsurface, resulting in surface passivation.

[0199] All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. The final thickness of the silicon dioxide can be selected toprovide the desired degree of electrical isolation in the device, wherea thicker layer of silicon dioxide provides a greater resistance toelectrical breakdown.

[0200] Photolithography and reactive-ion etching limit the layout designof separation post diameters and inter-post spacing to greater thanapproximately 1 μm. However, because the thermal oxidation processconsumes approximately 0.44 μm of silicon to form each micrometer ofsilicon dioxide, the thermal oxidation process results in a volumetricexpansion. This volumetric expansion may be utilized to reduce thespacing between the separation posts 416 to sub-micrometer dimensions.For example, with a layout inter-post spacing of approximately 1.5 μm,oxidation producing a 1 μm silicon dioxide film or layer would result ina nearest-neighbor spacing of approximately 0.5 μm. Further, because theoxidation process is well-controlled, separation post dimensions,including the inter-post spacing, in the sub-micrometer regime can beformed reproducibly and in a high yielding manner.

[0201]FIGS. 44A, 44B and 44C show scanning electron microscopephotographs and design layout of portions of fabricated liquidchromatography devices. FIG. 44A shows a design layout of a portion of areservoir and separation posts in a portion of a separation channelwhere the separation posts have rectangular cross-sectional shape. FIG.44B shows separation posts in a portion of a separation channel, theseparation posts having a circular cross-sectional shape and a diameterand inter-post spacing of approximately 1 μm. FIG. 44C shows separationposts in a portion of a separation channel, the separation posts havinga rectangular or square cross-sectional shape with a dimension of 2 μmand inter-post spacing of approximately 1 μm.

[0202] In a variation, the entrance orifice and the introduction channelfor filling the fluid reservoir may be formed in the cover substrate 524after a layer of silicon dioxide 525 is grown on all surfaces of thecover substrate 524, rather than in the substrate 500. As shown in FIG.45, the cover substrate 524 may be bonded to the reservoir side 503 ofthe separation substrate 500. The entrance orifice 406′ and theintroduction channel 404′ may be formed in the cover substrate 524 afteralignment with respect to the reservoir 410. The entrance orifice 406′and the introduction channel 404′ may be formed in the same or similarmanner as described above by utilizing lithography to define theentrance orifice pattern and reactive-ion etching to create the entranceorifice and the through-cover introduction channel. The cover substrate524 is again subjected to elevated temperature in an oxidizing ambientto grow a layer of oxide on the surface of the introduction channel404′. Further, the introduction channel 404′ may be formed from one ortwo sides of the cover substrate 524. If channel 404′ is formed from twosides of the cover substrate, the cover substrate 524 may be bonded tosubstrate 500 after forming the channel 404′ and after oxidation of thechannel surface. One advantage of defining the entrance orifice on thesame side of the completed liquid chromatography device as the reservoirand separation channel is that the back side of the substrate 500 isthen free from any features and may then be bonded to a protectivepackage without special provision for filling the reservoir through anentrance orifice defined on the back-side of the substrate.

[0203] Metallization for Fluid Flow Control

[0204]FIGS. 46A and 46B illustrate the formation of a reservoir, afilling, and an exit electrode as well as conductive lines or wiresconnecting the elctrodes to bond pads in the cover substrate 526.preferably comprising glass and/or silicon. The cover substrate 526shown in FIGS. 46A and 46B does not provide an entrance orifice or anintroduction channel although the metallization process described hereinmay be easily adapted for a cover substrate providing an entranceorifice and an introduction channel.

[0205] As shown in the plan and cross-sectional view of FIGS. 46A and46B, respectively, prior to the depositing of conductive material on thecover substrate 526, all surfaces of the cover substrate 526 aresubjected to thermal oxidization in a manner that is the same as orsimilar to the process described above to create a film or layer ofsilicon dioxide 528. Such oxidization is not performed where the coversubstrate 526 comprises glass.

[0206] The silicon dioxide layer 528 provides a surface on whichconductive electrodes may be formed. The thickness of the silicondioxide layer 528 is controllable through the oxidation temperature andtime and the final thickness can be selected to provide the desireddegree of electrical isolation, where a thicker layer of silicon dioxideprovides a greater resistance to electrical breakdown. The silicondioxide layer 528 electrically isolates all electrodes from the coversubstrate 526 and isolates the fluid in the reservoir and the channel ofthe liquid chromatography device from the cover substrate 526. Theability to isolate the fluid from the cover substrate 526 complementsthe electrical isolation provided in the separation substrate throughoxidation and ensures the complete electrical isolation of the fluidfrom both the separation substrate and the cover substrate 526. Thecomplete electrical isolation of the sample fluid from both substratesallows for the application of electric potential differences betweenspatially separated locations in the fluidic flow path resulting incontrol of the fluid flow through the path.

[0207] The cover substrate 528 may be cleaned after oxidation utilizingan oxidizing solution such as an actively oxidizing chemical bath, forexample, sulfuric acid (H₂SO₄) activated with hydrogen peroxide (H₂O₂).The cover substrate 528 is then thoroughly rinsed to eliminate organiccontaminants and particulates. A layer of conductive material 530 suchas aluminum is then deposited by any suitable method such as by DCmagnetron sputtering in an argon ambient. The thickness of the aluminumis preferably approximately 3000 Å, although shown having a largerthickness for clarity. Although aluminum is utilized in the fabricationsequence described herein, any type of highly conductive material suchas other metals, metallic multi-layers, silicides, conductive polymers,and conductive ceramics like indium tin oxide (ITO) may be utilized forthe electrodes. The surface preparation for satisfactory adhesion mayvary depending on the specific electrode material used. For example, thesilicon dioxide layer 528 provides a surface to which aluminumelectrodes may adhere as aluminum does not generally adhere well tonative silicon.

[0208] A film of positive-working photoresist 532 is then deposited overthe surface of the conductive material 530. Areas of the photoresistlayer 532 corresponding to areas surrounding the electrodes (shown) andconductive lines or wires and bond pads which will be subsequentlyetched are selectively exposed through a mask by an optical lithographicexposure tool passing short-wavelength light, such as blue ornear-ultraviolet at wavelengths of 365, 405, or 436 nanometers.

[0209] After development of the photoresist 532, the exposed areas ofthe photoresist are removed, leaving opening to the underlying aluminumconductive layer 530 while the unexposed areas 534, 536, 538corresponding to the reservoir, filling and exit electrodes,respectively, as well as conductive lines or wires and bond pads remainprotected by the photoresist. The conductive electrodes and thelines/bond pads may be etched, such as by a wet chemical etch or areactive-ion etch, as appropriate for the particular conductivematerial. The etch is selective to the underlying silicon dioxide layer528 or is terminated upon reaching the silicon dioxide layer 528 asdetermined by the etch time and rate. The remaining photoresist isremoved in an oxygen plasma or in a solvent bath such as acetone. Thefabrication sequence thus results in physically and electricallyseparate islands of conductive electrodes, lines and bond pads accordingto the pattern designed in the mask.

[0210] The cover substrate may be larger than the separation substrateto allow access to the bond pads and/or directly to the electrodes forthe application of potential voltage(s) to the electrode(s). As shown inFIG. 46C, the cover substrate 526′ is larger than the separationsubstrate such that the separation substrate only extends to dashed line540 relative to the cover substrate 526′. Conductive leadthroughs suchas connecting metal lines 542, 544 and 546 extend from the reservoir,filling and exit electrodes, 534, 536, 538, respectively, and enable theapplication of potential voltage(s) to the electrode(s).

[0211] Alternatively, a metal lead may be formed from each electrode toan otherwise unpatterned area of the separation substrate such that athrough-substrate access channel formed in the cover substrate andfilled with a conductive material by chemical vapor deposition (CVD)allows access to the electrode(s). As an alternative to chemical vapordeposition, the sidewalls of the through-substrate access channel may besloped, for example by KOH etch, to facilitate continuous deposition ofa conductive material thereon, thereby providing an electricallycontinuous path from the separation substrate to the top of the coversubstrate where potential voltages can be applied. In these variations,the separation and the cover substrates may be of the same size.

[0212] Although the electrodes are preferably provided on a surface ofthe cover substrate, the electrodes may be alternatively and/oradditionally provided on the separation substrate by appropriatemodifications to the above-described fabrication process. For example,in such a variation, the side walls of the reservoir are preferably notat a 90° angle relative to the bottom wall and can be formed at least inpart by, for example, a wet chemical potassium hydroxide (KOH) etch. Thesloped reservoir side walls allow for the deposition of a conductivematerial thereon. In another variation, the electrodes may also beformed by a damascene process, known in the art of semiconductorfabrication. The damascene process provides the advantage of a planarsurface without the step up and step down surface topography presentedby a bond line or pad and thus facilitates the bonding of the separationand cover substrate, as described below.

[0213] The above described fabrication sequence for the liquidchromatography device may be easily adapted to and is applicable for thesimultaneous fabrication of a monolithic system comprising multipleliquid chromatography devices including multiple reservoirs and/ormultiple separation channels as described above embodied in a singlemonolithic substrate.

[0214] Further, although the fabrication sequence is described in termsof fabricating a single liquid chromatography device, the fabricationsequence facilitates and allows for massively parallel processing ofsimilar devices. The multiple liquid chromatography devices or systemsfabricated by massively parallel processing on a single wafer may thenbe cut or otherwise separated into multiple devices or systems.

[0215] Although control of the liquid chromatography device has beendescribed above as comprising reservoir, filling and exit electrodes,any suitable combination of such and/or other electrodes in electricalcontact with the fluid in the fluid path may be provided and easilyfabricated by modifying the layout design. Further, any or all of theelectrodes may be additionally or alternatively provided in theseparation substrate. Electrodes may be formed in the separationsubstrate by modifying the fabrication sequence to include additionalsteps similar to or the same as the steps as described above withrespect to the formation of the electrodes in the cover substrate.

[0216] Bonding Cover Substrate to Separation Substrate

[0217] As described above, the cover substrate is preferablyhermetically bonded by any suitable method to the separation substratefor containment and isolation of the fluid in the liquid chromatographydevice. Examples of bonding silicon to silicon or glass to siliconinclude anodic bonding, sodium silicate bonding, eutectic bonding, andfusion bonding.

[0218] For example, to bond the separation substrate to a glass coversubstrate by anodic bonding, the separation substrate and coversubstrate are heated to approximately 400° C. and a voltage of 400-1200Volts is applied, with the separation substrate chosen as the anode (thehigher potential). Further, as the required bonding voltage depends onthe surface oxide thickness, it may be desirable to remove the oxidefilm or layer from the back side 505 of the separation substrate priorto the bonding process in order to reduce the required bonding voltage.The oxide film or layer may be removed by, for example, an unpatternedetch in a fluorine-based plasma. The etch is continued until the entireoxide layer has been removed, and the degree of over-etch isunimportant. Thus, the etch is easily controlled and high-yielding.

[0219] Critical considerations in any of the bonding methods include thealignment of features in the separation and the cover substrates toensure proper functioning of the liquid chromatography device afterbonding and the provision in layout design for conductive lead-throughssuch as the bond pads and/or metal lines so that the electrodes (if any)are accessible from outside the liquid chromatography device. Anothercritical consideration is the topography created through the fabricationsequence which may compromise the ability of the bonding method tohermetically seal the separation and cover substrates. For example, thestep up and step down in the surface topography presented by a metalline or pad may be particularly difficult to form a seal therearound asthe silicon or glass does not readily deform to conform to the shape ofthe metal line or pad, leaving a void near the interface between themetal and the oxide.

[0220] Integration of Liquid Chromatography and Electrospray Devices ona Chip

[0221] The cross-sectional schematic view of FIG. 47 shows a liquidchromatography-electrospray system 600 comprising a liquidchromatography device 602 of the present invention integrated with anelectrospray device 620 of the present invention such that a homogeneousinterface is formed between the exit orifice 614 of the liquidchromatography device 602 and the entrance orifice 622 of theelectrospray device 620. The single integrated system 600 allows for thefluid exiting the exit orifice 614 of the liquid chromatography device602 to be delivered on-chip to the entrance orifice 622 of theelectrospray device 620 in order to generate an electrospray.

[0222] As shown in FIG. 47, the entrance orifice 606 and theintroduction channel 604 of the liquid chromatography device 602 areformed in the cover substrate 608 along with the electrospray device620. Alternatively, the liquid chromatography entrance orifice and theintroduction channel may be formed in the separation substrate.

[0223] Fluid at the electrospray nozzle entrance 622 is at the exitvoltage applied to the exit electrode 610 in the separation channel 612near the liquid chromatography exit orifice 614. Thus, an electrosprayentrance electrode is not necessary.

[0224] The single integrated system 600 provides the advantage ofminimizing or eliminating extra fluid volume to reduce the risk ofundesired fluid changes, such as by reactions and/or mixing. The singleintegrated system 600 also provides the advantage of eliminating theneed for unreliable handling and attachment of components at themicroscopic level and of minimizing or eliminating fluid leakage bycontaining the fluid within one integrated system.

[0225] The integrated liquid chromatography-electrospray system 600 maybe utilized to deliver liquid samples to the sampling orifice of a massspectrometer. The sampling orifice of the mass spectrometer may serve asan extraction electrode in the electrospray process when held at anappropriate voltage relative to the voltage of the electrospray nozzle624. The liquid chromatography-electrospray system 600 may be positionedwithin 10 mm of the sampling orifice of the mass spectrometer forefficient extraction of the fluid from the electrospray nozzle 624.

[0226] Multiple Liquid Chromatography-electrospray Systems on a SingleChip Multiples of the liquid chromatography-electrospray system 600 maybe formed on a single chip to deliver a multiplicity of samples to acommon point for subsequent sequential analysis. For example, FIG. 48shows a plan view of multiple liquid chromatography-electrospray systems600 on a single chip 650 and FIG. 49 shows a detailed view of area A ofsystems 600 with the separation channels shown in phantom and withoutthe recessed portions for purposes of clarity. As shown, the multiplenozzles 624 of the electrospray devices 620 may be radially positionedabout a circle having a relatively small diameter near the center of thesingle chip 650. The dimensions of the electrospray nozzles and theliquid chromatography channels limit the radius at which multiplenozzles are positioned on the multi-system chip 650. For example, themulti-system chip may provide 96 nozzles with widths of up to 50 μmpositioned around a circle 2 mm in diameter such that the spacingbetween each pair of nozzles is approximately 65 μm.

[0227] Alternatively, an array of multiple electrospray devices withoutliquid chromatography devices may be formed on a single chip to delivera multiplicity of samples to a common point for subsequent sequentialanalysis. The nozzles may be similarly radially positioned about acircle having a relatively small diameter near the center of the chip.The array of electrospray devices on a single microchip may beintegrated upstream with multiple fluid delivery devices such asseparation devices fabricated on a single microchip. For example, anarray of radially distributed exit orifices of a radially distributedarray of micro liquid chromatography columns may be integrated withradially distributed entrance orifices of electrospray devices such thatthe nozzles are arranged at a small radius near the orifice of a massspectrometer. Thus, the electrospray devices may be utilized for rapidsequential analysis of multiple sample fluids. However, depending uponthe specific application and/or the capabilities of the downstream massspectrometer (or other downstream device), the multiples of theelectrospray devices may be utilized one at a time or simultaneously,either all or a portion of the electrospray devices, to generate one ormore electrosprays. In other words, the multiples of the electrospraydevices may be operated in parallel, staggered or individually.

[0228] The single multi-system chip 650 may be fabricated entirely insilicon substrates, thereby taking advantage of well-developed siliconprocessing techniques described above. Such processing techniques allowthe single multi-system chip 650 to be fabricated in a cost-effectivemanner, resulting in a cost performance that is consistent with use as adisposable device to eliminate cross-sample contamination. Furthermore,because the dimensions and positions of the liquidchromatographyelectrospray systems are determined through layout designrather than through processing, the layout design may be easily adaptedto fabricate multiple liquid chromatography-electrospray systems on asingle chip.

[0229] Interface of a Multi-system Chip to Mass Spectrometer

[0230] The radially distributed array of electrospray nozzles 624 on amulti-system chip may be interfaced with a sampling orifice of a massspectrometer by positioning the nozzles near the sampling orifice. Thetight radial configuration of the electrospray nozzles 624 allows thepositioning thereof in close proximity to the sampling orifice of a massspectrometer.

[0231] The multi-system chip 650 may be rotated relative to the samplingorifice to position one or more of the nozzles for electrospray near thesampling orifice. Appropriate voltage(s) may then be applied to the oneor more of the nozzles for electrospray. Alternatively, the multi-systemchip 650 may be fixed relative to the sampling orifice of a massspectrometer such that all nozzles, which converge in a relatively tightradius, are appropriately positioned for the electrospray process. As isevident, eliminating the need for nozzle repositioning allows for highlyreproducible and quick alignment of the single multi-system chip andincreases the speed of the analyses.

[0232] One, some or all of the radially distributed nozzles 624 of theelectrospray devices 620 may generate electrosprays simultaneously,sequentially or randomly as controlled by the voltages applied to theappropriate electrodes of the electrospray device 620.

[0233] While specific and preferred embodiments of the invention havebeen described and illustrated herein, it will be appreciated thatmodifications can be made without departing from the spirit of theinvention as found in the appended claims.

What is claimed is:
 1. An integrated miniaturized system for chemicalanalysis of fluids, comprising: an electrospray substrate having aninjection side and an ejection surface, the substrate defining anentrance orifice on the injection side, a nozzle on the ejectionsurface, a channel extending between the entrance orifice and thenozzle, and a region surrounding the nozzle recessed from the ejectionsurface; and electrode means for providing electrical contact to thefluids.
 2. The system of claim 1, wherein said electrode means comprisesan external conductor in contact with the fluid prior to said injectionside.
 3. The system of claim 1, wherein the channel has across-sectional area less than approximately 50,000 μm².
 4. The systemof claim 1, wherein the substrate defines a plurality of entranceorifices on the injection side, a plurality of nozzles on the ejectionsurface each corresponding to one of the plurality of entrance orifices,a plurality of channels each extending between one of the plurality ofnozzles and the corresponding one of the plurality of entrance orifices.5. The system of claim 4, wherein an array of said plurality of nozzlesare radially positioned on the ejection surface of the electrospraysubstrate.
 6. The system of claim 1, further comprising a device influid communication with the entrance orifice.
 7. The system of claim 1,wherein an array of nozzles are defined on the ejection surface of theelectrospray substrate, and further comprising a daughter plate defininga plurality of receiving wells positioned to receive a fluid ejectedthrough the nozzles of the electrospray substrate.
 8. The system ofclaim 1, further comprising a second substrate defining an entranceopening on a first surface and an exit on a second surface, the secondsubstrate being bonded to the electrospray substrate such that thesecond substrate exit is in fluid communication with the electrospraysubstrate entrance orifice.
 9. The system of claim 1, further comprisinga second substrate defining an entrance opening on a first surface, anexit on a second surface, a fluid reservoir recessed from the secondsurface, a separation channel recessed from the second surface, theseparation channel including the exit and extending between thereservoir and the exit, an introduction channel extending between theentrance opening and the reservoir, and a plurality of posts extendingfrom the separation channel, wherein the second substrate is bonded tothe electrospray substrate to enclose the reservoir and the separationchannel adjacent the electrospray substrate and such that the secondsubstrate exit is in fluid communication with the electrospray substrateentrance orifice.
 10. An electrospray device comprising: an array ofnozzles integrated in a substrate for ejecting a plurality of analytesat a mass spectrometry device interface; and a plurality of electrodesfor the application of electric potentials for generating andcontrolling an electric field at each nozzle to direct the ejection ofthe analytes from the nozzles within an acceptance region of the massspectrometry device.
 11. A method for generating an electrospray of afluid, comprising: providing a channel extending between an entranceorifice defined on an injection surface of a substrate and a nozzledefined on an ejection surface of the substrate; introducing a fluidinto the channel through the entrance orifice; providing a firstelectrode in electrical contact with the fluid; applying a firstpotential voltage to the fluid; positioning the nozzle adjacent to anextracting electrode; and ejecting the fluid from the channel throughthe nozzle by applying or holding the extracting electrode at a secondpotential voltage different from the first potential voltage.
 12. Themethod of claim 11, further comprising: providing a second electrode inelectrical contact with the substrate; and applying a third potentialvoltage to said second electrode, different from said first potentialvoltage.
 13. A liquid chromatography system, comprising: a firstsubstrate having a first surface and a second surface, the firstsubstrate defining an entrance opening on the first surface, a fluidreservoir recessed from the second surface, a first channel extendingbetween the entrance opening and the reservoir, a second channelrecessed from the second surface, and a plurality of posts extendingfrom the second channel; and a cover substrate bonded to the firstsubstrate to enclose the reservoir and the second channel adjacent thecover substrate, wherein the first and/or the cover substrate defines anexit and wherein the second channel extends between the exit and thereservoir.
 14. The liquid chromatography system of claim 13, furthercomprising an insulating layer provided over the surfaces of theseparation channel and the plurality of posts.
 15. The liquidchromatography system of claim 13, wherein the posts are spaced apartfrom each other by no more than 5 μm.
 16. The liquid chromatographysystem of claim 13, wherein the first substrate defines a plurality ofentrance openings on the first surface, a plurality of reservoirsrecessed from the second surface each corresponding to one of theplurality of entrance openings, one or more first channels eachcorresponding to and extending between one of the plurality of entranceopenings and the corresponding reservoir, and a plurality of secondchannels recessed from the second surface, wherein the first and/or thecover substrate defines a plurality of exits each corresponding to oneof the plurality of reservoirs, and wherein each second channelcorresponds to and extends between one of the plurality of reservoirsand the corresponding exit.
 17. A chemical separation device comprising:a substrate defining a channel, a plurality of posts fabricated fromsaid substrate and extending from said channel, and a stationary phasebound to the posts, said posts providing interaction with an analyteintroduced into said channel for producing separation.
 18. An integratedchemical analysis system, comprising: a first substrate having a majorsurface; a second substrate bonded to or otherwise attached to saidfirst substrate; a liquid chromatography system integrated in saidsecond substrate, and configured to receive a fluid for analysis and toprocess and output the fluid; and an electrospray device integrated onsaid first substrate, the electrospray device having an injectionsurface configured to receive the processed fluid from the liquidchromatography system, the major surface being configured to dispensethe fluid by electrospraying the fluid.
 19. The system of claim 18,wherein the major surface of the electrospray device is configured todispense the processed fluid in a direction generally perpendicular tothe major surface.
 20. The system of claim 18, wherein a plurality ofliquid chromatography systems are each integrated in said secondsubstrate, and each configured to receive a fluid for analysis and toprocess and output the fluid; and a plurality of electrospray devices,each integrated on said first substrate, the electrospray devices eachhaving an injection surface configured to receive the processed fluidfrom the liquid chromatography system, and each having an ejectionsurface configured to dispense the fluid by electrospraying the fluid.21. A system comprising: a microfabricated device defining a liquidchromatography device comprising an entrance for receiving an analyte,the device further defining an electrospray device including a nozzle,the electrospray device being configured to receive the analyte from theliquid chromatography device and to generate an electrospray; and a massspectrometer comprising a sampling orifice, said microfabricated devicebeing positioned to eject the electrospray from the nozzle into thesampling orifice.
 22. A method of mass spectrometric analysis utilizingan integrated chemical analysis device comprising: a firstmicrofabricated structure defining a liquid chromatography devicecomprising an entrance for receiving an analyte and an exit; and asecond microfabricated structure defining an electrospray deviceincluding an entrance for receiving the analyte from the liquidchromatography device and a nozzle in fluid communication with theentrance, the electrospray device to generate an electrospray andwherein the electrospray nozzle is adapted to eject the electrosprayfrom the nozzle into a sampling orifice of a mass spectrometer.